1 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT JIANLONG WANG Laboratory of Environmental Technology, INET, Tsinghua University, Beijing, China 1.1 INTRODUCTION In this chapter we offer an overview of the fundamentals and applications of biological processes developed for wastewater treatment, including aerobic and anaerobic processes. Beginning here, readers may learn how biosolids are even- tually produced during the biological treatment of wastewater. The fundamentals of biological treatment introduced in the first six sections of this chapter include (1) an overview of biological wastewater treatment, (2) the classification of microorgan- isms, (3) some important microorganisms in wastewater treatment, (4) measurement of microbial biomass, (5) microbial nutrition, and (6) microbial metabolism. Following the presentation of fundamentals, the remaining four sections introduce applications of biological wastewater treatment, including the functions of waste- water treatment, the activated sludge process, the suspended and attached-growth processes, and sludge production, treatment, and disposal. The topics covered include (1) aerobic biological oxidation, (2) biological nitrification and denitrifi- cation, (3) anaerobic biological oxidation, (4) biological phosphorus removal, (5) biological removal of toxic organic compounds and heavy metals, and (6) bio- logical removal of pathogens and parasites. Biological Sludge Minimization and Biomaterials/Bioenergy Recovery Technologies, First Edition. Edited by Etienne Paul and Yu Liu. Ó 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc. 1 COPYRIGHTED MATERIAL
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1FUNDAMENTALS OF BIOLOGICALPROCESSES FOR WASTEWATERTREATMENT
JIANLONG WANG
Laboratory of Environmental Technology, INET, Tsinghua University, Beijing, China
1.1 INTRODUCTION
In this chapter we offer an overview of the fundamentals and applications of
biological processes developed for wastewater treatment, including aerobic and
anaerobic processes. Beginning here, readers may learn how biosolids are even-
tually produced during the biological treatment of wastewater. The fundamentals of
biological treatment introduced in the first six sections of this chapter include (1) an
overview of biological wastewater treatment, (2) the classification of microorgan-
isms, (3) some importantmicroorganisms inwastewater treatment, (4)measurement
of microbial biomass, (5) microbial nutrition, and (6) microbial metabolism.
Following the presentation of fundamentals, the remaining four sections introduce
applications of biological wastewater treatment, including the functions of waste-
water treatment, the activated sludge process, the suspended and attached-growth
processes, and sludge production, treatment, and disposal. The topics covered
include (1) aerobic biological oxidation, (2) biological nitrification and denitrifi-
(5) biological removal of toxic organic compounds and heavy metals, and (6) bio-
logical removal of pathogens and parasites.
Biological Sludge Minimization and Biomaterials/Bioenergy Recovery Technologies, First Edition.Edited by Etienne Paul and Yu Liu.� 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.
1
COPYRIG
HTED M
ATERIAL
1.2 OVERVIEW OF BIOLOGICALWASTEWATER TREATMENT
Biological treatment processes are the most important unit operations in wastewater
treatment. Methods of purification in biological treatment units are similar to the self-
purification process that occurs naturally in rivers and streams, and involve many of
the same microorganisms. Removal of organic matter is carried out by heterotrophic
microorganisms, which are predominately bacteria but also, occasionally, fungi. The
microorganisms break down the organic matter by two distinct processes, biological
oxidation and biosynthesis, both of which result in the removal of organic matter
from wastewater. Oxidation or respiration results in the formation of mineralized end
products that remain in wastewater and are discharged in the final effluent, while
biosynthesis converts the colloidal and soluble organic matter into particulate
biomass (new microbial cells) which can subsequently be separated from the treated
liquid by gravity sedimentation because it has a specific gravity slightly greater than
that of water.
The fundamental mechanisms involved in biological treatment are the same for all
processes. Microorganisms, principally bacteria, utilize the organic and inorganic
matter present in wastewater to support growth. A portion of materials is oxidized,
and the energy released is used to convert the remainingmaterials into new cell tissue.
The aim in this chapter will help students and technicians in environmental science
and engineering to recognize the role that environmental microbiology plays in
solving environmental problems. The principal purposes of this chapter are (1) to
provide fundamental information on themicroorganisms used to treat wastewater and
(2) to introduce the application of biological process fundamentals for the biological
treatment of wastewater.
1.2.1 The Objective of Biological Wastewater Treatment
From a chemical point of view, municipal wastewater or sewage contains (1) organic
compounds, such as carbohydrates, proteins, and fats; (2) nitrogen, principally in the
form of ammonia; and (3) phosphorus, which is principally in the form of phosphate
from human waste and detergents. In addition, municipal wastewater contains many
other types of particulate and dissolvedmatter, such as pathogens, plastics, sand, grit,
live organisms, metals, anions, and cations. All these constituents have to be dealt
with at wastewater treatment plants. However, not all of these are important for the
modeling and design of a wastewater treatment plant. Usually, the carbonaceous,
nitrogenous, and phosphorus constituents are mainly objects to be considered
because they influence biological activity and eutrophication in the receiving water.
Whenmunicipal wastewater is discharged to awater body, the organic compounds
will stimulate the growth of the heterotrophic organisms, causing a reduction in the
dissolved oxygen. When oxygen is present, the ammonia, which is toxic to many
higher life forms, such as fish and insects, will be converted to nitrate by the nitrifying
microorganisms, resulting in a further demand for oxygen. Depending on the volume
of wastewater discharged and the amount of oxygen available, the water body can
become anoxic. If the water body does become anoxic, nitrification of ammonia to
2 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
nitrate by the autotrophic bacteria will cease. However, some of the heterotrophic
bacteria will use nitrate instead of oxygen as a terminal electron acceptor and
continue their metabolic reactions. Depending on the relative amount of organics and
nitrate, the nitrate may become depleted. In this case, the water will become
anaerobic and transfer to fermentation. When the organic compounds of the
wastewater have been depleted, the water body will begin to recover, clarify, and
again becomes aerobic. But most of the nutrients, nitrogen (N) and phosphorus (P),
remain and stimulate aquatic plants such as algae to grow. Only when the nutrients N
and P are depleted and the organic compounds sufficiently reduced can the water
body became eutrophically stable again.
From these considerations, the overall objectives of the biological treatment of
domestic wastewater are to (1) transform (i.e., oxidize) dissolved and particulate
biodegradable constituents into acceptable end products that will no longer sustain
heterotrophic growth; (2) transform or remove nutrients, such as ammonia, nitrate,
and particularly phosphates; and (3) in some cases, remove specific trace organic
constituents and compounds. For industrial wastewater treatment, 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 industrial wastewater can be
discharged to a municipal collection system.
1.2.2 Roles of Microorganisms in Wastewater Treatment
The biological wastewater treatment is carried out by a diversified group of
microorganisms. It is the bacteria that are primarily responsible for the oxidation
of organic compounds. However, fungi, algae, protozoans, and higher organisms all
have important roles in the transformation of soluble and colloidal organic pollutants
into carbon dioxide and water as well as biomass. The latter can be removed from the
liquid by settlement prior to discharge to a natural watercourse.
Many water pollution problems and solutions deal with microorganisms. To solve
water pollution problems, environmental scientists require a background in micro-
biology. By understanding how microbes live and grow in an environment, envi-
ronmental engineers can develop the best possible solution to biological waste
problems. After construction of the desired treatment facilities, environmental
engineers are responsible to operate them properly to produce the desired results
at the least cost.
Learning to use mixtures of microorganisms to control the major environmental
systems has become a major challenge for environmental engineers. Environmental
microbiology should help operators of municipal wastewater and industrial waste
treatment plants gain a better understanding of how their biological treatment unit
work and what should be done to obtain maximum treatment efficiency. Design
engineers should gain a better understanding of how microbes provide the desired
treatment and the limitations for good design. Even regulatory personnel can obtain a
better understanding of the limits of their regulations and what concentration of
contaminants can be allowed in the environment.
OVERVIEW OF BIOLOGICAL WASTEWATER TREATMENT 3
The stabilization of organic matter is accomplished biologically using a variety of
microorganisms, which convert the colloidal and dissolved carbonaceous organic
matter into various gases and into protoplasm. It is important to note that unless the
protoplasm produced from the organic matter is removed from the solution, complete
treatment will not be accomplished because the protoplasm, which itself is organic,
will be measured as biological oxygen demand (BOD) in the effluent.
1.2.3 Types of Biological Wastewater Treatment Processes
Biological treatment processes are typically divided into two categories according
to the existing state of the microorganisms: suspended-growth systems and
attached-growth systems. Suspended systems are more commonly referred to as
activated sludge processes, of which several variations and modifications exist.
Attached-growth systems differ from suspended-growth systems in that microor-
ganisms attached themselves to a medium, which provides an inert support.
Trickling filters and rotating biological contactors are most common forms of
attached-growth systems.
The major biological processes used for wastewater treatment are typically
divided into four groups: aerobic processes, anoxic processes, anaerobic processes,
and a combination of aerobic/anoxic or anaerobic processes. The individual pro-
cesses are further subdivided into two categories: suspended-growth systems and
attached-growth systems, according to the existing state of microorganisms in the
wastewater treatment systems.
1.3 CLASSIFICATION OF MICROORGANISMS
Conventional taxonomic methods used to identify a bacterium rely on physical
properties of the bacteria and metabolic characteristics. To apply this approach, a
pure culture must first be isolated. The culture may be isolated by serial dilution and
growth in selective growth media. The cells are harvested and grown as pure culture
using sterilization techniques to prevent contamination. Historically, the types of
tests that are used to characterize a pure culture include (1) microscopic observations,
to determine morphology (size and shape); (2) Gram staining technique; (3) the type
of electron acceptor used in oxidation–reduction reactions; (4) the type of carbon
source used for cell growth; (5) the ability to use various nitrogen and sulfur sources;
(Plasmodium malaria); (4) Ciliophora: ciliated protozoans (Paramecium). The first
three classes are the free-swimming protozoans and the last class comprises the
parasitic protozoans. The full scheme is illustrated in Fig. 1.3.
Protozoans are primarily aerobic organisms, requiring dissolved oxygen as their
electron acceptor. Although protozoans can be grown in concentrated, complex
nutrient media, they prefer to use bacteria as their source of nutrients. The protozoans
metabolize the biodegradable portion of the bacteria for energy and synthesis and
excrete the nonbiodegradable fraction back into the environment. Although the
majority of protozoans are aerobic organisms, there are anaerobic protozoans. Like
their bacteria counterparts, the anaerobic protozoans must eat tremendous quantities
of nutrients to obtain sufficient energy for cell synthesis. The low bacteria growth in
anaerobic environments means that anaerobic protozoans will be found only in high-
organic-concentration environments.
Protozoans undergo reproduction by fission, splitting into two cells along the
longitudinal axis. It takes several hours for the two cells to split completely. Growth
Sporozoa
Sarcodina
Protozoa
Sub-Kingdom Phylum Subphylum
MastigophoraPhytomastigophora
Zoomastigophora
Autotracta
Hydraula
Piroplasma
Apicomplexa
Myxospora
Microspora
Kinetofragminophora
Oligohymenophora
Polyhymenophora
Ciliophora
FIGURE 1.3 Classification of protozoans.
SOME IMPORTANT MICROORGANISMS IN WASTEWATER TREATMENT 17
continues as long as environmental conditions are favorable. When environmental
conditions begin to turn bad for continued growth of the protozoans, they form cysts.
Each cyst is produced by coating the nucleus with a hard shell, allowing the nucleus to
survive in adverse environments. The rest of the cell tissues become nutrients for
additional bacteria growth.When the cyst finds a reasonable environment for growth,
the nucleus begins to expand, creating new protozoans.
Environmental factors such as pH and temperature have the same relative effect on
protozoans as on bacteria. Protozoans grow best at pH levels between 6.5 and 8.5.
Strongly acidic or strongly alkaline conditions are toxic to protozoans. As far as
temperature is concerned, protozoans can be either mesophilic or thermophilic, the
same as bacteria. Most protozoans are mesophilic, having a maximum temperature
for growth of around 40�C. Protozoans change their rate of metabolism by a factor of
2 for each 10�C temperature change, the same as the other organisms. Protozoans
have difficulty surviving at temperatures below 5�C because the viscosity of thewater
increases, making it more difficult for the protozoans to move and obtain food.
1.4.5 Rotifers and Crustaceans
Both rotifers and crustaceans are animals: aerobic multicellular chemoheterotrophs.
The rotifer derives its name from the apparent rotating motion of two sets of cilia on
its head. The cilia provide mobility and a mechanism for catching food. Rotifers
consume bacteria and small particles of organic matter. Crustaceans, a group that
includes shrimp, lobsters, and barnacles, are characterized by their shell structure.
They are a source of food for fish and are not found in wastewater treatment systems
to any extent except in underloaded lagoons. Their presence is indicative of a high
level of dissolved oxygen and a very low level of organic matter.
Rotifers are multicellular, microscopic animals with flexible bodies. They are
larger than protozoans and have complex metabolic systems. Like the other micro-
scopic animals, rotifers prefer bacteria as their source of food, but can eat small algae
and protozoans. The rotifers have cilia around their mouths to assist in gathering
food. The cilia also provide motility for the rotifers if they do not remain attached to
solid particles with their forked tails. The flexible bodies allow the rotifers to bend
around and feed on bacteria and algae attached to solid surfaces. A typical rotifer is
shown in Fig. 1.4.
Philodina is one of the most common rotifers. The cilia give the appearance of two
rotating wheels at the head of the rotifer.Epiphanes is a large rotifer, reaching 600 mmin length. Some rotifers are as small as 100 mm.Rotifers are all strict aerobes andmust
FIGURE 1.4 Schematic diagram of a typical rotifer.
18 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
have several micrograms per liter of dissolved oxygen in order to grow. They can
survive for several hours in low dissolved oxygen (DO) environments, but not for
long periods. In the presence of large bacteria populations and adequate DO, rotifers
will quickly eat most of the bacteria, even if the bacteria are flocculated. In a suitable
environment the rotifers can quickly metabolize all the bacteria and then starve to
death. Excessive growth of rotifers can be controlled by reducing the dissolved
oxygen to prevent them from growing so rapidly. The DO can be reduced to around
1.0mgL�1 to favor the metabolism of aerobic bacteria and protozoans and slow the
growth of rotifers. As large complex organisms, rotifers require lots of bacteria in
their growth. Rotifers can remove the bacteria attached to solid surfaces and can
ingest small, flocculated masses of bacteria. They are more sensitive to environ-
mental stresses than either bacteria or protozoans. Temperature affects rotifers in the
sameway that temperature affects the othermicroorganisms. Their metabolism slows
as the temperature decreases and increases as the temperature rises. There do not
appear to be any thermophilic rotifers. Reproduction in rotifers occurs through egg
formation rather than by binary fission. Rotifer eggs can remain dormant for a
considerable period of time if environmental conditions are not satisfactory for
growth. It has been difficult to study the quantitative growth characteristics of rotifers
since they cannot be grown free of bacteria.
Rotifers play an important role in the overall food chain from bacteria and algae to
higher organisms. They are widely found in the aquatic environment, where there is a
suitable environment for growth. Rivers, lakes, and reserviors are good sources of
rotifers. The environments that favor rotifers tend to favor other higher animal forms.
Crustaceans are multicellular animals with hard shells to protect their bodies.
They also have jointed appendages attached to their bodies. The appendages assist in
movement and food gathering. The large size of the crustaceans, 1.5 to 2mm, makes
them visible to the naked eye if one looks very carefully. Being more complex than
rotifers, they growmore slowly and are more sensitive to environmental changes. The
crustaceans feed on bacteria, algae, protozoans, and solid organic materials.
Daphnia and Cyclops are two common crustaceans. They are easily found in
freshwater lakes in the warm summer months. They require high levels of DO and a
moderate level of nutrients. It has been estimated that Daphnia require about 80% of
their body weight each day for maximum growth. Only about 20% of the food
consumed ends up as cell mass. The larger mass of the Daphnia requires a
considerable number of smaller organisms to remain alive and to grow. Since the
Daphnia are relatively large, they become food for macroscopic organisms in the
water environment.
The presence or absence of sufficient concentrations of trace metals in the bacteria
or algae used as their food source also affects the magnitude of growth of the different
species of crustaceans. The U.S. Environmental Protection Agency (EPA) has
proposed the use of Ceridaphnia as the indicator organism for effluent toxicity
from wastewater treatment plants. Unfortunately, Ceridaphnia is a very sensitive
crustacean that can be difficult to maintain in the laboratory for routine use. Research-
ers are currently examining otherDaphnia in an effort to find a suitable crustacean that
is both sensitive to toxic substances and easy to maintain in the laboratory.
SOME IMPORTANT MICROORGANISMS IN WASTEWATER TREATMENT 19
1.4.6 Viruses
Viruses belong neither to prokaryotes nor to eukaryotes. They carry out no catabolic
or anabolic function. Their replication occurs inside a host cell. The infected cells
may be animal or plant cells, bacteria, fungi, or algae. Viruses are very small (25 to
350 nm) and most of them can be observed only with an electron microscope.
A virus is made of a core of nucleic acid (double- or single-stranded DNA;
double- or single-stranded RNA) surrounded by a protein coat called a capsid.
Capsids are composed of arrangements of various numbers of protein subunits
known as capsomeres. The combination of capsid and nucleic acid core is called
nucleocapsid.
Bacterial phages have been used as models to elucidate the phases involved in
virus replication. The various phases are as follows (Fig. 1.5):
1. Adsorption and entry. This is the first step in the replication cycle of viruses. In
order to infect the host cells, the virus must adsorb to receptors located on the
cell surface. Animal viruses adsorb to surface components of the host cell.
The receptors may be polysaccharides, proteins, or lipoproteins. Then the virus
or its nucleic acid enters the host cell. Bacteriophages “inject” their nucleic
acid into the host cell. For animal viruses the entire virion penetrates the host
cell by endocytosis.
2. ENTRY
1. ADSORPTION
3. MULTIPLICATION 4. MATURATION
5. RELEASE
FIGURE 1.5 Viral lytic cycle.
20 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
2. Eclipse. During this step, the virus is “uncoated” (i.e., stripping of the capsid),
and the nucleic acid is liberated.
3. Replication. This step involves the replication of the viral nucleic acid.
4. Maturation and release. The protein coat is synthesized and is assembled with
the nucleic acid to form a nucleocapsid. Virus release is generally attributable
to the rupture of the host cell membrane.
1.5 MEASUREMENT OF MICROBIAL BIOMASS
Various approaches are available for measuring microbial biomass in laboratory
cultures or in environmental samples, as shown in Fig. 1.6.
1.5.1 Total Number of Microbial Cells
The total number of cells (live and dead cells) can be measured by using special
counting chambers such as the Petroff–Hauser chamber for bacterial counts or
Fermentation Amajor product in the catabolic pathways discussed above is that of
the reduced pyridine nucleotides. A cell contains only a very limited amount of
NADþ and NADPþ. For the central metabolic pathways of dissimilation to continue,
a means for continuously regenerating these from the reduced forms must exist.
Microorganisms do this either by fermentation or by respiration.
Fermentation is a metabolic process in which the reduced pyridine nucleotides
produced during glycolysis or other dissimilatory pathways are used to reduce an
organic electron acceptor that is synthesized by the cell itself (i.e., an endogenous
Citrate(6C)
CoA
CoACoA
CO2
CO2
CO2
H2O
H2O
H2O
H2O
NAD+
NAD+
NAD+
NAD+
NADH + H+
NADH + H+
NADH + H+
NADH + H+
(3C) Pyruvate
(6C) Cis-Aconitase
(6C) Isocitrate
(5C) α-Ketoglutarate
Oxaloacetate (4C)
Malate (4C)
Fumarate (4C)
Succinate (4C) (4C) Succinyl CoA
CoA
CoA
GTP GDP + Pi
FADH2
FAD
Pyruvate dehydrogenasecomplex
Aconitase
Aconitase
Isocitratedehydrogenase
α-Ketoglutaratedehydrogenase complex
Succinyl CoAsynthase
Succinatedehydrogenase
Fumarase
Malatedehydrogenase
Citrate synthase
(3C) Acetyl
FIGURE 1.11 The citric acid cycle.
36 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
electron acceptor). Many microbes utilize derivatives of pyruvate as electron and Hþ
acceptors, and this allows NAD(P)H þ Hþ to be reoxidized to NAD(P). For
example, when yeast cells are grown under anaerobiosis, they carry out an alcoholic
fermentation. After making pyruvate by glycolysis, they remove a molecule of CO2
from pyruvate to form acetaldehyde:
pyruvic acid! acetaldehydeþ CO2 ð1:4ÞThe acetaldehyde is the acceptor for the electrons of the NADH þ Hþ produced
during glycolysis and becomes reduced to ethanol, thus regenerating NADþ :
acetaldehydeþ NADHþ Hþ ! ethanolþ NADþ ð1:5Þ
Other microorganisms use different fermentations to regenerate NADþ . Lacticacid fermentation is a common type of fermentation characteristic of lactic acid
bacteria and some Bacillus species. For example Lactobacillus lactis carries out a
lactic acid fermentation by using pyruvic acid itself as the electron acceptor:
pyruvateþ NADHþ Hþ ! lactic acidþ NADþ ð1:6Þ
Just as the alcoholic fermentation is of great importance to the alcoholic beverage
industry, the lactic acid fermentation is important for the dairy industry. The many
other types of fermentations carried out by bacteria lead to various end products, such
as propionic acid, butyric acid, butylene glycol, isopropanol, and acetone. Fermen-
tation is an inefficient process for extracting energy by the cell because the end
products of fermentation still contain a great deal of chemical energy. For example,
the high energy content of the ethanol produced by yeasts is indicated by the fact that
ethanol is an excellent fuel and liberates much heat when burned.
In summary, fermentation is the transformation of pyruvic acid to various products
in the absence of a terminal electron acceptor. Both the electron donor and acceptor
are organic compounds and ATP is generated solely via substrate-level phosphor-
ylation. Fermentation releases little energy (2 ATP per molecule of glucose), and
most of it remains in fermentation products. The latter depends on the type of
microorganism involved in fermentation.
Respiration Respiration is the other process for regenerating NAD(P)þ by using
NAD(P)H as the electron donor for an electron transport system. It is much more
efficient than fermentation for yielding energy. Not only is NAD(P)þ regenerated,
but the electron transport system generates a proton motive force that can be used to
power the synthesis of additional ATP molecules. For example, when yeast cells are
grown aerobically with glucose, the NADH molecules produced during glycolysis
can donate their electrons to an electron transport system that has oxygen as the
terminal electron acceptor (aerobic respiration). This system allows not only
regeneration of NADþ but also the generation of enough of a proton motive
force to drive the synthesis of an additional six molecules of ATP. Further breakdown
occurs when pyruvic acid is oxidized to acetyl-CoA by pyruvate dehydrogenase.
MICROBIAL METABOLISM 37
Each of the two molecules of NADH so formed can serve as the electron donor for an
electron transport system, creating a proton motive force that can be used for
synthesizing six molecules of ATP. Oxidation of the acetyl-CoA by the citric acid
cycle yields six more NADH molecules, which can be used to make 18 ATP. In
addition, two molecules of FADH2 are produced that can provide enough energy for
the synthesis of four molecules of ATP. Two additional ATPs are made by substrate-
level phosphorylation. Therefore, the net yield of ATP from complete dissimilation of
one glucose molecule is 38 molecules (Fig. 1.12). This is in sharp contrast to the yield
of ATP from fermentation when yeast cells are grown anaerobically, where the yield
is only two molecules of ATP per molecule of glucose. The complete breakdown of
glucose to six molecules of CO2 results in a net yield of 38 ATP molecules.
1.7.2 Anabolic Metabolic Pathway
Anabolism (biosynthesis) includes all the energy-consuming processes that result in
the formation of new cells. It is estimated that 3000mmol of ATP is required to make
100mg of dry mass of cells. Moreover, most of this energy is used for protein
FIGURE 1.12 ATP production by aerobic respiration.
38 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
synthesis (Brock and Madigan, 1991). Cells use energy (ATP) to make building
blocks, synthesize macromolecules, repair damage to cells (maintenance energy),
and maintain movement and active transport across the cell membrane. Most of the
ATP generated by catabolic reactions is used for biosynthesis of biological macro-
molecules such as proteins, lipids, polysaccharides, purines, and pyrimidines. Most
of the precursors of these macromolecules (amino acids, fatty acids, monosacchar-
ides, nucleotides) are derived from intermediates formed during glycolysis, the Krebs
cycle, and other metabolic pathways (Entner–Doudoroff and pentose phosphate
pathways). These precursors are linked together by specific bonds (e.g., peptide bond
for proteins, glycoside bond for polysaccharides, phosphodiester bond for nucleic
acids) to form cell biopolymers. This is the pathway by which organisms synthesize
protoplasm (construct new cell mass). A fraction of the organic molecules taken up
by the organism is modified enzymatically to form part of the biological protoplasm.
This synthesis process requires an input not only of organic molecules, but also
inorganic molecules [e.g., ammonia (NH4), phosphorus, and micronutrients], energy,
protons, and electrons. The organic components of organismmass are highly complex
and very numerous, but principally are proteins, fats (lipids), and carbohydrates.
The formation of these compounds (anabolism) follows a wide variety of biochemical
pathways too complex to trace.
The primary purpose of the energy reaction is to provide the energy for cell
synthesis. The bacteria must synthesize hundreds of different chemical compounds to
make a cell. It is not surprising to find that the bacteria use the same basic chemical
structures repeatedly, greatly simplifying the synthesis of new cells. It is too difficult to
analyze every compound produced in each cell; but it is possible to use total
measurements of the organic fraction of bacteria for the synthesis reactions. The
primary method for evaluation of synthesis has been to develop an empirical equation
of the cell mass from the carbon, hydrogen, oxygen, and nitrogen analyses of bacteria.
The techniques for evaluating the empirical formula of bacteria protoplasm have
already been presented. It has been found that the bacteria produce the same cell
protoplasm regardless of the chemical nature of the substrate, as long as all nutrients are
available for normal cell production. The nutrients and the environment determine the
overall synthesis reaction. The energy in the cell mass must come from the substrate
being metabolized. Since the heat of combustion determination for bacterial cell mass
oxidizes nitrogen to nitrogen gas, the heat of combustion values measured are high
compared to the energy requiredby the bacteria to formaunitmass of protoplasm.Most
bacteria use ammonia nitrogen as their source of cell nitrogen. Only a few specialized
nitrogen-fixing bacteria are able to use nitrogen gas as their source of cell nitrogen. The
total energy requirement for the synthesis of bacteria protoplasmwill be the energy for
synthesis of the cell components plus the energy content of the cell mass produced.
1.7.3 Biomass Synthesis Yields
In the evaluation and modeling of biological treatment systems, we should distin-
guish the yield observed and the synthesis yield (or true yield). The biomass yield
observed is based on the actual measurements of biomass production and substrate
MICROBIAL METABOLISM 39
consumption, which are actually less than the synthesis yield because of cell loss
concurrent with cell growth.
In full-scale wastewater treatment processes the term solids production (or solids
yield) is also used to describe the amount of VSS generated in the treatment process.
The term is different from the synthesis biomass yield values, because it contains
other organic solids from the wastewater that are measured as VSS, but they are not
biological solids. 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 measured 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 following factors: (1) the energy that can be derived from the oxidation–reduction
reaction; (2) the growth characteristics of the carbon source; (3) the nitrogen source;
and (4) environmental factors such as temperature, pH, and osmotic pressure. The
synthesis yield can be estimated if the stoichiometry or the amount of energy
produced in the oxidation–reduction reaction is known.
The activated sludge process removes substrate, which exerts an oxygen demand
by converting the food into new cell material and degrading this substrate while
generating energy. This cell material ultimately becomes sludge, which must be
disposed of. Despite the problems in doing so, researchers have attempted to develop
enough basic information on sludge production to permit a reliable design basis. A
net yield of 0.5 kg mixed liquor VSS (MLVSS) kg�1 BOD5 removed could be
expected for a completely soluble organic substrate. Most researchers agree that
depending on the inert solids in the system and the sludge retention time, 0.40 to
0.60 kg MLVSS kg�1 BOD5 removed will normally be observed.
The amount of sludge that must be wasted each day is the difference between the
amount of increase in sludge mass and the suspended solids (SS) lost in the effluent.
mass to be wasted ¼ increase in MLSS� SS lost in effluent ð1:7Þ
The net activated sludge produced each day is determined by
Yobs ¼ Y
1þ kdycð1:8Þ
Px ¼ Yobs Q ðS0 � SÞ ðkg=1000 gÞ ð1:9Þ
where Px is the netwaste activated sludge produced each day in terms ofVSS (kg d�1),
Yobs the yield observed (kgMLVSS kg�1 BOD5 removed), Q the wastewater flow rate
(m3 d�1), S the soluble BOD5 in aeration tank and effluent (mgL�1), kd the decay rate
of microorganisms (d�1), and yc the mean cell residence time (d).
The increase in MLSS may be estimated by assuming that VSS is some fraction
of MLSS. It is generally assumed that VSS is 60 to 80% of MLVSS. Thus, the
increase in MLSS in Eq. (1.9) may be estimated by dividing Px by a factor of 0.6 to
0.8 (or multiplying by 1.25 to 1.667). Themass of suspended solids lost in the effluent
is the product of the flow rate (Q� Qw) and the suspended solids concentration (Xe).
40 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
Typical synthesis yield coefficients are given in Table 1.7 for common electron
donors and acceptors in wastewater treatment.
1.7.4 Coupling Energy-Synthesis Metabolism
The bacteria couple the energy-synthesis reactions into a single set of reactions
rather than in separate reactions. Substrate metabolism results in a continuous
processing of nutrients to create energy for the manufacture of cell mass and the cell
mass produced from the use of that synthesis energy. Since the cell mass contains the
same amount of energy per unit weight, the energy requirement to produce that unit
of cell mass is also constant. It was reported that 62% of the substrate energy was
conserved as a cell mass, with 38% being used to produce the energy for synthesis.
The data on cell mass yields, based on glucosemetabolism, have shownvariations in
the conversion of glucose and ammonia to cell mass energy ranging from 50 to 70%.
A major part of the problem with glucose metabolism is the production of organic
storage products in addition to the production of cell mass. Another part of the
variation in energy-synthesis relationships can be traced to analytical problems in
harvesting and weighing small quantities of bacteria. Still, another part of the
problem is related to maintenance energy expended during the growth period.
Maintenance energy, also termed endogenous respiration, is the energy used to
maintain cell integrity and depends on the active bacteria mass, time and temper-
ature. The longer the growth period for the bacteria, the greater is the effect of
endogenous respiration. The net effect of endogenous respiration is to reduce the
proteins contained in the new cell mass.
Close coupling of the energy synthesis–endogenous respiration reactions allows
the energy from the endogenous respiration reaction to pass without notice until
studies are carried out over long periods under substrate-limiting conditions. Since
most bacteriological studies are carried out in excess substrate over short growth
periods, endogenous respiration is masked. Temperature also affects endogenous
respiration. Endogenous respiration increases as the temperature of metabolism
increases. For this reason endogenous respiration has a greater impact on bacteria
grown at 37�C than on bacteria grown at 20�C. Additional research is needed on
evaluation of the impact of endogenous respiration by bacteria.
Based on 62% energy conversion to cellmass, normalmetabolism requires a total of
31.6 kJ g�1 cellmass produced. The energy requirements for cell synthesis are the same
TABLE 1.7 Typical Bacteria Synthesis Yield Coefficients for Common Biological
Reactions in Wastewater Treatment
Growth Condition Electron Donor Electron Acceptor Synthesis Yield
Aerobic Organic compound Oxygen 0.40 g VSS g�1 COD
Aerobic Ammonia Oxygen 0.12 g VSS g�1 NH4-N
Anoxic Organic compound Nitrate 0.30 g VSS g�1 COD
Anaerobic Organic compound Organic compound 0.06 g VSS g�1 COD
Anaerobic Acetate Carbon dioxide 0.05 g VSS g�1 COD
MICROBIAL METABOLISM 41
for aerobic metabolism as for anaerobic metabolism. The cell mass contains the same
amount of energy, and the synthesis reactions require the same amount of energy to
produce a unit of cell mass. The difference between aerobic metabolism and anaerobic
metabolism lies in the amount of energyproducedunder the two environments.Aerobic
metabolismproducesmore energy thananaerobicmetabolism from the sameamount of
substratemetabolized. The net effect is thatmore cellmass is produced aerobically than
ylene 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.
A number of chlorinated organic compounds are degradable by cometabolic
degradation, including trichloroethene, dichloroethene, vinyl chloride, chloro-
form, dichloromethane, and trichloroethane. Cometabolic degradation is possible
by bacteria that produce nonspecific monooxygenase or dioxygenase enzymes.
These enzymes mediate a reaction with oxygen and hydrogen and change the
structure of the chlorinated compound. The reaction of the nonspecific oxygenase
enzyme with the organic chlorinated compound typically produces an interme-
diate compound that is degraded by other aerobic heterotrophic bacteria in the
biological consortia.
Removal of Heavy Metals Heavy metals sources in wastewater treatment plants
include mainly industrial discharges and urban stormwater runoff. Toxic metals may
adversely affect biological treatment processes as well as the quality of receiving
waters. They are inhibitory to both anaerobic and aerobic processes in wastewater
treatment. The removal of heavy metals in biological treatment processes is mainly
by adsorption and complexation through the interaction between the metals and the
microorganisms. In addition, transformations and precipitation of metal ions are also
possible to occur.Microorganisms combinewith heavymetal ions and adsorb them to
cell surfaces because of interactions between the metal ions and the negatively
charged microbial surfaces. Metal ions may also be complexed by the carboxyl
groups in microbial polysaccharides and other polymers, or absorbed by protein
materials in the biological cell.
A significant amount of soluble metal removal has been observed in biological
processes (activated sludge, trickling filter, oxidation ponds), with removal efficiency
ranging from 50 to 98%, depending on the initial metal concentration, the biological
reactor solids concentrations, and the system sludge retention time (SRT). In anaerobic
processes the reduction of sulfate to hydrogen sulfide can promote the precipitation of
metal ions as sulfides. For example, adding ferric or ferrous chloride to anaerobic
digesters can remove sulfide toxicity by forming iron sulfide precipitates.
FUNCTIONS OF BIOLOGICAL WASTEWATER TREATMENT 57
In general, the removal of heavy metals by microorganisms may be due to
solubilization, precipitation, chelation, biomethylation, or volatilization. The heavy
metals may be removed for the following reasons: (1) production of strong acids,
such as H2SO4, by chemoautotrophic bacteria (e.g., Thiobacillus), which dissolve
minerals; (2) production of organic acids (e.g., citric acid), which not only dissolve
but also chelate metals to form metal–organic molecules; (3) production of ammonia
or organic bases, which precipitate heavy metals as hydroxides; (4) extracellular
metal precipitation (e.g., sulfate-reducing bacteria produce H2S, which precipitates
heavy metals as insoluble sulfides); (5) production of extracellular polysaccharides,
which can chelate heavy metals and thus reduce their toxicity; (6) bacteria (e.g.,
sheathed filamentous bacteria) that fix Fe and Mn on their surface in the form of
hydroxides or some other insoluble metal salts; and (7) biotransformation by bacteria
that have the ability to biomethlylate or volatilize (e.g., Hg), oxidize (e.g., As), or
reduce (e.g., Cr) heavy metals.
1.8.5 Removal of Pathogens and Parasites
Pathogens and parasites can be removed and/or inactivated in aeration and
sedimentation tanks of the activated sludge process. During the aeration phase,
environmental (e.g., temperature, sunlight) and biological (e.g., inactivation by
antagonistic microorganisms) factors, possibly aeration, have an impact on
pathogen and parasite survival. Floc formation during the aeration phase is
also instrumental in removing undesirable microorganisms. During the sedimen-
tation phase, certain microorganisms (e.g., parasites) undergo sedimentation,
while floc-entrapped microbial pathogens settle readily in the tank. As compared
with other biological treatment processes, activated sludge is relatively efficient in
removing pathogenic microorganisms and parasites from incoming primary
effluents.
1. Bacteria. Activated sludge is generally more efficient than trickling filters for
the removal of indicator (e.g., coliforms) and pathogenic (e.g., Salmonella)
bacteria. The removal efficiency may vary from 80% to more than 99%.
Bacteria are removed through inactivation, grazing by ciliated protozoa, and
adsorption to sludge solids or encapsulation within sludge flocs, or both,
followed by sedimentation.
2. Viruses. The activated sludge process is the most efficient biological process
for virus removal from sewage. It appears that most of the virus particles are
solids-associated and are ultimately transferred to sludge. The ability of
activated sludge to remove viruses is related to the capacity to remove solids.
Thus, many of the viruses found in the effluents are solids-associated. Viruses
are also inactivated by environmental and biological factors.
In summary, virus removal and inactivation by activated sludge may be due to the
following: (1) virus adsorption to or encapsulation within sludge solids (this results in
the transfer of viruses to sludge); (2) virus inactivation by sewage bacteria (some
58 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
activated sludge bacteria may have some antiviral activity); or (3) virus ingestion by
protozoans (ciliates) and small metazoans (e.g., nematodes).
Protozoan cysts, such as Entamoeba histolytica and Giardia cysts, are not
inactivated in the aeration tank of an activated sludge process. They are, however,
entrapped in sludge flocs and are thus transferred to sludge after sedimentation.
Under laboratory conditions, the activated sludge process removes 80 to 84% of
Cryptosporidium parvum oocysts. In-plant reduction of Cryptosporidium by the
activated sludge process varied from 84.6 to 96.8%. The removal efficiency was
higher for Giardia than for Cryptosporidium. However, the removal of protozoan
parasites by biological treatment is very variable. The removal efficiency was
estimated at 0 to 90% for Cryptosporidium and 60 to 90% for Giardia cysts in a
wastewater treatment plant that incorporates both primary and secondary stages.
Because of their size and density, eggs of helminth parasites (e.g., Taenia,
Ancylostoma, Necator) are removed by sedimentation during primary treatment
of wastewater and during the activated sludge treatment; thus, they are largely
concentrated in sludge.
1.9 ACTIVATED SLUDGE PROCESS
The activated sludge process is the most popular process for treating both domestic
and industrial wastewater. It has been used around the world for almost a century, yet
is still very much a “black box.” Some of the major chemical changes that are taking
place can readily be measured (Fig. 1.18), but still, little is known about the microbes
that are responsible for these and how their activities are affected by such variables as
influent composition, process configuration, and process operation. The application
of molecular methods to the activated sludge ecosystem has not only revealed a
previously unsuspected level of microbial diversity, but has also questioned many
FIGURE 1.18 Main chemical transformations that occur in the activated sludge process.
ACTIVATED SLUDGE PROCESS 59
earlier ideas as to which bacteria were responsible for important processes such as
phosphorus removal.
1.9.1 Basic Process
The most widely used biological treatment processes is the activated sludge process,
which was developed in England in 1913. It was so named because it involved the
production of an activated mass of microorganisms capable of aerobically stabilizing
a waste. Many versions of the original processes are in use today, but fundamentally,
they are all similar.
Activated sludge is an aerobic, suspended growth system. Activated sludge
treatment plants use circular, square, or rectangular aeration tanks. The aeration
tank provides a suitable environment for a mixture of bacteria and other micro-
organisms to aerobically metabolize the biodegradable contaminants in the incoming
wastewater. Oxygen is supplied to the microbes by diffused air, mechanical surface
aerators, submerged turbines, or impingement jets. The ability of activated sludge to
flocculate under quiescent conditions and to separate from the treated wastewaters is
an essential characteristic of the activated sludge process. The keys to activated
sludge are excess microorganisms, excess dissolved oxygen, sufficient time, and
adequate mixing in the aeration tank to promote rapid metabolism of the biode-
gradable organic compounds. Excess microorganisms can be maintained in the
aeration tank as a result of the ability of bacteria to form floc after the nutrients have
been metabolized and to settle quickly by gravity under quiescent conditions
before being collected and pumped back to the aeration tank as return activated
sludge. The continuous stabilization of organic matter in wastewater results in the
production of more microorganisms than are needed to maintain the activated sludge
at its desired concentration in the aeration tank. The extra microbial production has
been termed excess activated sludge. The excess sludge must be removed from the
activated sludge system to maintain the desired microbial population. A small
amount of excess sludge will be lost in the final effluent, about 10 to 30mgL�1.
Most of the excess sludge must be wasted from the system as waste activated sludge.
The basic activated sludge process is shown in Fig. 1.19. Although there have been
numerous variations in activated sludge systems, the basic process for activated
sludge systems has not changed.
EffluentFINALSEDTANK
AERATION TANK
Waste Activated Sludge
Return Activated Sludge
Air
Influent
FIGURE 1.19 Schematic diagram of the activated sludge process.
60 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
The activated sludge in the aeration tank is called mixed liquor suspended solids.
The settled sludge returned to the aeration tank is called return activated sludge and
the sludge removed for wasting is called waste activated sludge. The suspended
solids carried out in the final effluent are called effluent suspended solids.
1.9.2 Microbiology of Activated Sludge
Buswell and Long reported their studies on activated sludge microbiology in 1923.
They used daily microscopic examinations of the mixed liquor to obtain gross
information on activated sludge organisms. Most of their data dealt with protozoans,
since they were easily counted under the microscope. They found that the initial
population of small flagellated protozoans and small ciliated protozoans quickly gave
way to larger free-swimming ciliated protozoans and stalked ciliated protozoans. As
the system became more stable, crawling ciliated protozoans appeared. Only a few
rotifers were observed. Nematodes suddenly appeared and then slowly decreased.
The overall bacteria were simply classified as zoogleal masses with filamentous
bacteria of various types. They thought that both bacteria and protozoans were
important in activated sludge.
Bacteria The overall environment in the aeration tank determines which micro-
organisms grow and to what extent they grow. An optimum environment for bacteria
includes pH between 7.0 and 8.0; temperature between 20 and 30�C; dissolved oxygenabove1.0mgL�1 at all times in all parts of the aeration tank, sufficient agitation to keep
the suspended solids in uniform suspension; a readily biodegradable substrate with
adequate carbon, nitrogen, phosphorus, and trace nutrients; and adequate time for
complete metabolism. The bacteria best able to metabolize the substrate and produce
new cell masswill automatically predominate. Normalwastewater load variationswill
allow the bacteria population to adjust to the changing organic loads. The predominant
bacteria will be motile rod-shaped bacteria that can metabolize the maximum amount
of organic contaminants to the greatest extent.
All types of aerobic bacteria will grow to the extent that they can obtain nutrients.
If the environment is changed, the bacteria predomination will change. It is important
to recognize that the only way to change the bacteria predomination in a given
activated sludge system is to change the environment in the aeration tank.
Activated sludge treating municipal wastewaters will have bacteria acclimated to
proteins, carbohydrates, and lipids. The relative population of acclimated bacteria
will depend upon the rate of addition of the three groups of organic compounds. The
treatment of unusual organic compounds in industrial wastewaters can pose a
challenge in accumulating the desired acclimated bacteria for efficient treatment.
In addition to providing a good environment in the aeration tank, it is important to
maintain the specific bacteria necessary for metabolism of the industrial
contaminants.
The bacteria in activated sludge are capable of metabolizing most organic
compounds found in nature. The few complex organic compounds that cannot be
metabolized in a reasonable time period are considered to be nonbiodegradable.
ACTIVATED SLUDGE PROCESS 61
Lignin and bacteria polysaccharides are two complex organic compounds that cannot
be metabolized in activated sludge systems in a reasonable period of time.
The growth of filamentous bacteria is normal in activated sludge and will not be a
problem unless the filamentous bacteria population increases sufficiently to adversely
affect the settling rate of the activated sludge. Excessive growth of filamentous
bacteria will cause activated sludge to bulk. Bulking sludge is light, fluffy, and slow to
flocculate and settle in the final sedimentation tanks. Filamentous bacteria are able to
predominate over the normal bacteria when oxygen is limiting, in high-carbohydrate
wastewaters, under nitrogen limitation, at low pH, and in excessively long SRT
systems. About 95% of the filamentous bulking problems in activated sludge are the
result of an inadequate oxygen supply during stabilization of the organic compounds
in wastewaters.
It has been proposed that an anaerobic selector be used to prevent the growth of
filamentous bacteria. With the anaerobic selector, the incoming wastewaters and the
return activated sludge are mixed without aeration to allow the facultative bacteria to
begin to grow under anaerobic conditions. If the filamentous bacteria are strict
aerobes, they will not grow and will continue to die off under anaerobic conditions.
The total aerobic bacterial counts in standard activated sludge are on the order of
108 CFU/mg of sludge. When using culture-based techniques, it was found that the
major genera in the flocs are Zooglea, Pseudomonas, Flavobacterium, Alcaligenes,
Achromobacter, Corynebacterium, Comomonas, Brevibacterium, Acinetobacter, and
Bacillus spp., as well as filamentous microorganisms. Some examples of filamentous
microorganisms are the sheathed bacteria (e.g., Sphaerotilus) and the gliding bacteria
(e.g., Beggiatoa, Vitreoscilla), which are responsible for sludge bulking. Table 1.9
displays some bacterial genera found in standard activated sludge using culture-based
techniques. The majority of the bacterial isolates were identified as Comamonas–
Pseudomonas species.
TABLE 1.9 Distribution of Aerobic Heterotrophic Bacteria in
Standard Activated Sludge
Genus or Group % Total Isolates
Comamonas–Pseudomonas 50.0
Alcaligenes 5.8
Pseudomonas (fluorescent group) 1.9
Paracoccus 11.5
Unidentified (gram-negative rods) 1.9
Aeromonas 1.9
Flavobacterium–Cytophaga 13.5
Bacillus 1.9
Micrococcus 1.9
Coryneform 5.8
Arthrobacter 1.9
Aureobacterium–Microbacterium 1.9
62 FUNDAMENTALS OF BIOLOGICAL PROCESSES FOR WASTEWATER TREATMENT
The cocci referred to as the “G-bacteria” and often seen in activated sludge
systems in large numbers are phylogenetically diverse and contain mainly novel
gram-positive and gram-negative bacteria. They are seen microscopically as tetrads
or aggregates in activated sludge. They dominate in systems with poor phosphorus
removal because they outcompete phosphorus-accumulating organisms by accumu-
lating polysaccharides instead of polyphosphates. Two strains of G-bacteria were
identified as Tetracoccus cechii and belong to the alpha group of proteobacteria.
The general understanding of their physiology is poor, so their role in EBPR plants is
still not certain. All seem to possess an ability to synthesize intracellular storage
polymers, but the conditions affecting their production await clarification. It is likely
that there are many more G-bacteria in activated sludge systems.
Zoogloea are exopolysaccharide-producing bacteria that produce typical finger-
like projections and are found in wastewater and other organically enriched
environments. These projections consist of aggregates of Zooglea cells surrounded
by a polysaccharide matrix. They are found in various stages of wastewater
treatment, but their numbers comprise only 0.1 to 1% of the total bacterial numbers
in the mixed liquor.
Activated sludge flocs also harbor autotrophic bacteria such as nitrifiers (e.g.,
Nitrosomonas, Nitrobacter), which convert ammonium to nitrate. Nitrifying bacteria
use carbon dioxide as their primary source of cell carbon. The growth of nitrifying
bacteria is much slower than the heterotrophic bacteria in activated sludge systems.
Although the only real competition between the nitrifying bacteria and the hetero-
trophic bacteria is for DO, the greater growth of heterotrophic bacteria limits the
ability of the nitrifying bacteria to obtain nutrients. Dense floc keeps the nitrifying
bacteria from the necessary nutrients for rapid growth. The complete nitrification
requires a SRT value in aeration of 3 to 5 days at 20�C.
Fungi Activated sludge does not usually favor the growth of fungi, although some
fungal filaments are observed in activated sludge flocs. Fungi may grow abundantly
under specific conditions of low pH, toxicity, and nitrogen-deficient wastewater. The
predominant genera found in activated sludge are Geotrichum, Penicillium, Cepha-
losporium, Cladosporium, and Alternaria. Sludge bulking may result from the
abundant growth of Geotrichum candidum, which is favored by low pH from
acid wastes. Fungi are also capable of carrying out nitrification and denitrification,
suggesting that they could play a role in nitrogen removal in wastewater under
appropriate conditions. Some advantages of a fungi-based treatment system are the
ability of fungi to carry out nitrification in a single step, and their greater resistance to
inhibitory compounds than bacteria.
Protozoans Protozoans are significant predators of bacteria in activated sludge as
well as in natural aquatic environments. Protozoans were observed regularly in
activated sludge, there was a clear succession of protozoans during the development
of activated sludge treating municipal wastewater. The initial growth of amoeboid
protozoans and small flagellated protozoans gave way to small free-swimming
ciliated protozoans and then, to larger free-swimming ciliated protozoans. Finally,
ACTIVATED SLUDGE PROCESS 63
stalked ciliated protozoans and crawling ciliated protozoans predominated as the
activated sludge reached normal operating conditions.
Ciliates appear to be the most abundant protozoans in activated sludge plants.
They are subdivided into free, creeping, and stalked ciliates. Free ciliates feed on
free-swimming bacteria. The most important genera found in activated sludge are