chapter 2 review of literature -...
TRANSCRIPT
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CHAPTER 2
Review of Literature
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2.1. Introduction to Wastewater Treatment
2.1.1. History and Composition of Domestic Wastewater
The practice of wastewater treatment started at the beginning of 20th century. The
activated sludge process was discovered by accident in Britain in 1913. Experiments on
treating sewage in a draw-and-fill reactor (the precursor to today's sequencing batch
reactor) produced a highly treated effluent. Believing that the sludge had been ‘activated”
the process was named activated sludge. The goal, as proposed by the British Royal
Commission on sewage disposal, was to produce final effluent of 30mg/L suspended
solids and 20mg/L of Biological Oxygen Demand-BOD (Sterrit and Lester, 1988).
Today, there are greater than 15000 wastewater treatment facilities in U.S alone.
Major contaminants in wastewater are biodegradable organics, volatile organics,
recalcitrant xenobiotics, toxic metals, suspended solids, nutrients (N, P), & microbial
pathogens and parasites (Figure 2.1). Objectives of wastewater treatment process are;
1. Reduction of the organic content of wastewater (Reduction of BOD)
2. Removal/reduction of trace organics that are recalcitrant to biodegradation and
may be toxic or carcinogenic
3. Removal/ reduction of toxic metals
4. Removal/ reduction of nutrients (N, P) to reduce pollution of surface or ground
water.
5. Removal or inactivation of pathogens and parasites.
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Figure 2.1: Contaminants of wastewater treatment system (Metcalf & Eddy, 1991)
Domestic wastewater is a combination of human and animal excreta (feces & urine) and
gray water resulting from washing, bathing and cooking. Bulk of organic matter in
domestic wastewater is easily biodegradable and consists mainly of carbohydrates, amino
acids, peptides and proteins, volatile acids, fatty acids and their esters (Giger & Roberts,
1978; Painter & Viney, 1959). The chemical characteristics of a typical untreated
domestic wastewater are presented in Table 2.1 (Metcalf & Eddy, 1991).
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Concentration
Parameters Strong (mg/L) Medium (mg/L) Weak (mg/L)
BOD5 400 220 110
COD 1000 500 250
Organic N 35 15 8
NH3-N 50 25 12
Total N 85 40 20
Total P 15 8 4
Total Solids 1200 720 350
Suspended
Solids
350 220 100
Table 2.1: Typical Characteristics of Domestic wastewater (Metcalf & Eddy, 1991)
2.1.2 Activated Sludge Process:
Activated sludge process (ASP), a secondary treatment process, is an important stage in
sewage treatment process which is considered to be a biological process and has found
vast application as an effective means of wastewater treatment (Gerhardi and Frank,
1990). Activated sludge is a process in sewage treatment in which air or oxygen is forced
into sewage liquor to develop a biological floc which reduces the organic content of the
sewage (Nutrient removal). In all activated sludge plants, once the sewage has received
sufficient treatment, excess mixed liquor is discharged into settling tanks and the
supernatant is run off to undergo further treatment before discharge. Part of the settled
material, the sludge, is returned to the aeration system to re-seed the new sewage entering
the tank. This fraction of the floc is called Return Activated Sludge (RAS). The
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remaining sludge, called Waste Activated Sludge (WAS), is further treated prior to
disposal. WAS is also sometimes called Surplus Activated Sludge (SAS).
Activated sludge is also the name given to the active biological material produced by
activated sludge plants and which affects all the purification processes. This material,
which in healthy sludge is a brown floc, is largely composed of saprophytic bacteria but
also has an important protozoan flora mainly composed of amoebae, Spirotrichs,
Peritrichs including Vorticellids and a range of other filter feeding species. Other
important constituents include motile and sedentary Rotifers.
Purpose
In a sewage treatment plant, Activated Sludge process can be used for one or several of
the following purposes:
1. Oxidizing carbonaceous matter: biological matter
2. Oxidizing nitrogenous matter: mainly ammonium and nitrogen in biological
materials.
3. Phosphate removal
4. Driving off entrained gases - carbon dioxide, ammonia, nitrogen etc.
5. Generating a biological floc that is easy to settle.
6. Generating a liquor low in dissolved or suspended material
i.e., in summary there are two main goals of an ASP:
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1. Oxidation of the biodegradable organic matter in the aeration tank (soluble
organic matter is thus converted to new cell mass)
2. Flocculation, that is, the separation of the newly formed biomass from the
treated effluent.
Basically, activated sludge comprises a microbiological enrichment culture consisting of
a mixed, and largely uncontrolled, consortium of micro- and macro-organisms known
collectively as activated sludge that remove soluble and insoluble wastewater organics
and converts this material into a flocculent microbial suspension that settles well in a
conventional gravity clarifier (Ramothokang et al., 2003). The activated sludge
microorganisms derive energy from carbonaceous organic matter in aerated wastewater
for the production of new cells in a synthetic process, while simultaneously releasing
energy through the conversion of this organic matter into compounds of lower energy,
such as carbon dioxide and water, in a process called respiration. The separation and
settling of activated-sludge solids is accomplished by creating an acceptable quality of
secondary wastewater effluent, and the collection and recycling of microorganisms back
into the system or removal of excess microorganisms from the system. Hence the
diversity of microbial community in the activated sludge plant depends on the influent
wastewater, environmental parameters such as pH and temperature and prevalent
operational conditions (Wilderer et al., 2002; Martins et al. 2004).
Some operational parameters commonly used in activated sludge systems are defined as
follows (Davis and Cornwell, 1985; Verstraete and van Vaerenbergh, 1986).
Mixed Liquor Suspended Solids (MLSS): The content of the aeration tank in an
activated sludge system. It is the total amount of organic and mineral suspended solids,
including microorganisms, in the mixed liquor.
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Mixed Liquor Volatile Suspended Solids (MLVSS): The organic portion of MLSS is
represented by MLVSS, which comprises non microbial organic matter, as well as dead
and live microorganisms and cellular debris (Nelson and Lawrence, 1980).
Food-to-Microorganism Ratio (F/M): The F:M ratio indicates the organic load into the
activated sludge system.
Hydraulic Retention Time (HRT): HRT is the average time spent by the influent liquid in
the aeration tank of the ASP. It is the reciprocal of the dilution rate.
Sludge Age: It is the mean residence time of microorganisms in the system. It is the
reciprocal of the microbial growth rate.
2.1.3 Components of ASP:
Conventional ASP comprises of the following:
Aeration tank: Aerobic oxidation of organic matter is carried out in this tank. Primary
effluent is introduced and mixed with return activated sludge (RAS) to form the mixed
liquor, which contains 1500-2500 mg/L of suspended solids. Aeration is provided by
mechanical means. An important characteristic of the activated sludge process is the
recycling of a large portion of the biomass. This makes the mean cell residence time (i.e.
sludge age) much greater than the hydraulic retention time (Sterritt and Lester, 1988).
This practice helps maintain a large number of microorganisms that effectively oxidize
organic compounds in a relatively short time. The detention time in the aeration basin
varies between 4 and 8 hours.
Sedimentation tank: This tank is used for the sedimentation of microbial flocs (sludge)
produced during the oxidation stage in the aeration tank. A portion of the sludge in the
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clarifier is recycled back to the aeration basin and the reminder is wasted to maintain a
proper F/M (Food to Microorganism ratio).
The Physical component (for removing carbonaceous pollution) of the activated-sludge
process contains five essential interrelated components (Activated Sludge, Manual of
Practice No.9 Water Environment Association,1987) :
a. Aeration tanks to introduce air or oxygen into the system to create an aerobic
environment and that keeps the activated sludge properly mixed
b. Aeration source pure oxygen, compressed air or mechanical aeration
c. Secondary Tanks or Secondary clarifiers allow the biological flocs to settle during
which activated-sludge solids separate from the surrounding waste water by the
process of flocculation and the return activated sludge sediment by gravity.
d. A system of pumps to pump back return activated sludge to the aeration tanks to
replenish the biological community.
e. A system of pumps to remove activated sludge containing an overabundance of
microorganisms. This is necessary in order to control the food-to-microorganism
ratio in the aeration tanks.
This is illustrated in the following diagram: (Figure 2.2; www.wikipedia.org)
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Figure 2.2: Generalized schematic showing flow of sewage in an activated sludge
system
Treatment of nitrogenous matter or phosphate involves additional steps where the mixed
liquor is left in anoxic condition (no residual dissolved oxygen).
The Biological component of the activated sludge system is comprised of
microorganisms. The composition of these microorganisms is 70 to 90 percent organic
matter and 10 to 30 percent inorganic matter.
Bacteria, fungi, protozoa, and rotifers constitute the biological component, or biological
mass, of activated sludge. A color atlas of wastewater organisms is available and
generally consulted to become familiar with the most encountered organisms in activated
sludge or trickling filters (Berk and Gunderson, 1993).
The species of microorganism that dominates a system depends on environmental
conditions, process design, the mode of plant operation, and the characteristics of the
secondary influent wastewater. (Water Environment Association, 1987) The
microorganisms that are of greatest numerical importance in activated sludge are aerobic
and anaerobic bacteria. An important factor under low dissolved oxygen concentrations is
that the bacteria living in activated sludge is facultative.
While both heterotrophic and autotrophic bacteria reside in activated sludge, the former
predominate. Important genera of heterotrophic bacteria include Achromobacter,
Alcaligenes, Arthrobacter, Citromonas, Flavobacterium, Pseudomonas, Comomonas,
Brevibacterium, Acinetobacter, Bacillus sp. and Zoogloea. (Jenkins et al, 1993). A gram
negative cocci known as 'G bacteria' are also found as tetrads in activated sludge
(Seviour, 2002). They dominate in system with poor phosphorous removal.
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Autotrophic bacteria in activated sludge reduce oxidized carbon compounds such as
carbon dioxide for cell growth. These bacteria obtain their energy by oxidizing ammonia
nitrogen to nitrate nitrogen in a two-stage conversion process known as nitrification. Due
to the fact that very little energy is derived from these oxidization reactions, and because
energy is required to convert carbon dioxide to cellular carbon, nitrifying bacteria
represent a small percentage of the total population of microorganisms in activated
sludge. In addition, autotrophic nitrifying bacteria have a slower rate of reproduction than
heterotrophic, carbon-removing bacteria. Two genera of bacteria are responsible for the
conversion of ammonia to nitrate in activated sludge, Nitrobacter and Nitrosomonas.
(Water Environment Society, 1987).
Other common microorganisms found in aerobic systems include Beggiatoa,
Geotirichum and Sphaerotilus. Protozoans (such as Vorticella, Opercularia, Epistylis)
and rotifers consume dispersed bacteria and small biological floc particles that have not
settled.
Suspended growth systems are composed of Bdellovibrio, Lecicothrix, Mycobacterium,
Nocardia, Thiothrix and Zoogloea bacteria while Alcaligenes, Chlorella, Fusarium,
Mucor, Penicillium, Phormicium, Sphaerotilus natans, Sporatichum, Ulothrix and Yeasts
make up the attached growth systems. Algae such as Chlorella, Phormicium, Ulothrix
are not directly involved in waste stabilization. They add oxygen to the system but they
can cause clogging of the system, which produces odors.
A notable population of animal and bacterial viruses are also found in wastewater;
particularly human viruses that are excreted in large quantities in feces. These viruses are
found to decrease quantitatively by the activated-sludge treatment process (Grabow,
1968).
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As a general rule, the nature of the wastewater will dictate the preferred process
modifications, primarily for the purpose of maintaining mixed liquor settling quality
(Eckenfelder and Musterman, 1995).
2.1.4. Role of Bacteria in the Activated Sludge Process
Bacteria, both aerobic and anaerobic, make up about 95% of the activated sludge biomass
and grow in wastewater by consuming biodegradable matter, by the help of enzymes.
Bacteria are comprised of proteins, carbohydrates, fats and many other compounds.
Bacteria use the food mostly for growth and the rest of it is converted to energy which is
very much required for various activities like motility and reproduction.
When there is very little food available, the bacteria use the limited food to produce
energy and to maintain the cell. Very little is available for growth so less reproduction
occurs. Under these circumstances bacteria loses it flagella and thus, its motility in an
attempt to conserve energy. The excreted waste products begin to form a thick slime
layer outside the cell wall, making the cells stick together.
The number of aerobic bacteria decreases as the floc size increases. The inner region of
relatively large floc favors strict anaerobes such as methanogens or sulfur reducers.
Bacteria, particularly the Gram negative, constitute the major component of activated
sludge flocs. Hundreds of bacterial strains thrives activated sludge but only a small
fraction can be detected by culture based techniques. They are responsible for the
oxidation of organic matter and nutrient transformations, and produce polysaccharides
and other polymeric compounds that aid in flocculation of biomass. However, by culture
based technique, less than 10% of total cell numbers obtained by direct microscopic
counting could be detected. New approaches for characterizing bacterial communities in
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activated sludge include 16s and 23s rRNA targeted oligonucleotide fluorescent probes
for in situ identification of bacteria (Manz et.al, 1994; Wagner et.al, 1993).
2.1.4.1 Food to Microorganism ratio (F/M)
The amount of biodegradable matter available as food for the bacteria is measured in
terms of BOD (biochemical oxygen demand) or COD (chemical oxygen demand) in the
influent sewage. The weight of microorganisms in the mixed liquor is estimated by
measuring the amount of volatile suspended solids (VSS) in the activated sludge. This
information is used to form a relationship called food to microorganism ratio (F/M ratio).
The F/M ratio gives an idea about the growth conditions of the cells. If the F/M ratio is
high, the bacteria normally grow quite rapidly (because this means there is a lot of "food"
available in comparison to the amount of microorganism); if the F/M ratio is low, the
bacteria normally grow very slowly (because little food is available for growth).
The F/M ratio is now considered as a process control number that helps to determine the
proper number of microorganisms for a given system. F/M ratio control ranges for typical
ASPs are given in Table 2.2.
Calculation of F: M ratio:
The term is actually a measurement of the amount of incoming food ( Lbs of Influent
CBOD) divided by the Lbs of Microorganisms in the system. F: M ratio is calculated
from the following values.
1. Influent Flow into your activated sludge system (Million Gallons per Day-MGD)
2. Influent CBOD (mg/l or ppm) concentration into your aeration tank.
3. Mixed Liquor Volatile Suspended Solids Concentration (mg/l)
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4. Volume (in gallons) of your aeration system
Realizing that 1 Gallon of water weighs 8.34 pounds
The weight of Food entering the process:
F in Lbs/day = Influent Flow (MGD) X Influent CBOD Concentration (mg/l) X 8.34
The weight of Microorganisms under aeration:
M in pounds= Aeration System Volume (in Millions of Gallons) X MLVSS X 8.34
The ratio F/M gives the Food to Microorganisms ratio.
The F:M ratio is expressed in Kilogram BOD per Kilogram MLSS per day (Curds and
Hawkes, 1983; Nathanson, 1986). The general formula for calculating F/M is as follows.
Q X BOD
F/M = ---------------
MLSS X V
Q = Flow rate of sewage in million gallons per day
BOD = 5 day Biochemical Oxygen Demand in mg/L
MLSS = Mixed Liquor Suspended Solids mg/L
V = Volume of aeration tank in gallons
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Table 2.2: F/M ratio control ranges for typical ASPs (Florida Water Resources Journal)
Process Range Names Common SWT ASP Names F/M Range
Extended Aeration
Extended Aeration
Sequencing Batch Reactors Race
Track or Orbital Ditch
0.05-0.15 Lb CBOD5/1 Lb
MLTSS
Standard Activated Sludge
Conventional Activated Sludge
Contact Stabilization
Step Aeration
Complete (or Homogenous) Mix
Others used with nutrient removal
0.25-0.5 Lb CBOD5/1 Lb
MLTSS
Hi-Rate Activated Sludge HRAS based on desired removal 1.0-10 Lb CBOD5/1 Lb
MLTSS
2.1.4.2 Oxygen uptake rate:
Actively growing microorganisms use oxygen at a rapid rate. The rate at which oxygen is
used is measured by a test called the Oxygen Uptake Rate (OUR), or the Respiration
Rate. It is measured in mg O2/hr/gm of MLSS. - Normally a higher uptake rate is
associated with high F/M ratios and younger sludges and a lower uptake rate is associated
with lower F/M and older sludges.
2.1.4.3 Formation of Flocs:
As the sludge is allowed to age, the bacteria lose their motility and accumulate more
slime. Then the clumps and chains formed by the bacteria are better able to stick together.
The clumps grow bigger and bigger until they form a floc. If the organisms are allowed to
develop properly, under the right conditions, the floc becomes large and compact and
begins to settle. The mixing in the aeration tank tends to keep the floc small since, even
though the bacterial cells are sticky, the bond formed holding the organisms together is
not very strong which is good as it improves the contact between the cells, food, and
oxygen.
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Further, the conditions which affect the health of the microorganisms and favor optimum
process conditions are Dissolved Oxygen (DO), Mixing, pH, Temperature and Nutrients.
Dissolved Oxygen (DO) levels
It is important to maintain about 2 mg/L of D.O. in the activated sludge so that the
bacteria that are contained in the floc are supplied with enough oxygen. If the DO
is less than 2 mg/L, the bacteria on the outside of the floc use up the DO before it
get into the center of the floc and may lead to cell death causing the floc to break
up.
Mixing
Proper mixing is required to bring in contact organisms, oxygen, and nutrients and
to remove metabolic waste products. Excessive mixing can cause floc break up or
unstable floc formation.
pH
The enzymes which regulate many of the biochemical reaction in bacteria are pH
dependent. The optimum pH should be maintained (between 7.0 and 7.5) for
proper activated sludge microorganisms to dominate.
Temperature
All the biochemical reactions taking place with and outside the cells are
temperature dependent. Lower temperatures cause such reactions to be much
slower. Thus, more number of bacteria are required to do the same job during the
winter than in the summer.
Nutrients
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Microorganisms require certain nutrients for growth. The basic nutrients of
abundance in normal raw sewage are carbon (C), nitrogen (N), phosphorus (P),
with the ratio of C:N:P ratio approximately equal to 100:10:1. In addition to
C,N,and P, trace amounts of sodium (Na), Potassium (K), magnesium (Mg), iron
(Fe), and many others are required. In municipal sewage, most of these nutrients
are provided. Most problems with nutrient deficiency occur when there are a lot
of industrial wastes present. When proper nutrients are not available, the
metabolism fails and a kind of bacterial fat (slime) will begin to accumulate
around the cell. The cells slow down in their activity and shall not settle properly
which leads to improper BOD removal.
2.1.4 Filamentous Bacteria
Over growth of filamentous bacteria poses two critical problems, filamentous bulking and
foaming, in any sewage treatment plant. These two problems influence significantly on
the treatment efficiency. So far according to Eikelboom (1975), twenty-six types of
filaments were identified and grouped into seven groups (Table.2.3)
Group I Sheath forming, Gram –ve bacteria
Sphaerotilus natans, Type 1701, Type 1702
Haliscomenobacter hydrosis and Type 0321
Group II Sheath forming, Gram +ve bacteria
Type 0041, Type 0675, Type 1851
Group III Sheathless curled, multicellular bacteria
resembling blue-green algae
Type 021N, Nostocoida limicola, Cyanophyceae
Group IV Slender coiled bacteria
Microthrix parvicella, Type 0581, Type 0192
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Group V Straight, multicellular, Gram –ve bacteria
Type 0803, Type 1091, Type 0091, Type 0961
Group VI Gliding filamentous bacteria
Type 0914, Beggiatoa spp., Type 1111, Type
1501
Group VII Additional types
Type 1863, Type 0411, Fungi and Nocardia spp.
Table 2.3: Groups of Filamentous Organisms occurring in ASP
a. Filamentous Bulking:
Filamentous bulking is caused by excessive growth of filamentous bacteria,
significantly effecting settling and compaction properties of the sludge . A
bulking sludge is defined as one that settles and compacts slowly and shows a
sludge volume index (SVI) of >150 ml/g. However, each plant has a specific SVI
value depending on the treatment plant's ability to contain the sludge within the
final clarifier which in turn is dependent on the size and performance of the final
clarifier(s) and hydraulic considerations.
A certain amount of filamentous bacteria is beneficial to the activated sludge
process. Absence of filamentous bacteria can lead to small, easily sheared flocs
(pin-floc) that settle well but leave behind a turbid effluent.
Filaments serve as a "backbone" to floc structure, allowing the formation of
larger, stronger flocs. The presence of some filaments also serves to catch and
hold small particles during sludge settling, yielding a lower turbidity effluent.
Filamentous bulking is the number one cause of effluent noncompliance today.
The causative filaments, their identification system and control of filamentous
bulking and foaming is extensively discussed by Eikelboom and van Buijsen
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(Eikelboom and van Buijsen, 1975 & 1981) which is later updated and modified
by Jenkins et al. (1993, 2003) and is being followed worldwide. Once the
causative filaments are identified, their causes could be determined and control
measures appropriate to each filament can be found.
Depending on the type of filament involved, two forms of interference in sludge
settling occur: (1) interfloc-bridging - where the filaments extend from the floc
surface and physically hold the floc particles apart; and (2) open-floc structure -
where the filaments grow mostly within the floc and the floc grows around and
attached to the filaments. Here, the floc becomes large, irregularly-shaped, and
contains substantial internal voids. A bulking sludge can result in the loss of
sludge inventory to the effluent, causing environmental damage and effluent
violations. In severe cases, loss of the sludge inventory can lead to a loss of the
plant's treatment capacity and failure of the process. Additionally, disinfection of
the treated wastewater can become compromised by the excess solids present
during bulking. In less severe cases, bulking leads to excessive return sludge
recycle rates and problems in waste activated sludge disposal. Many problems in
waste sludge thickening are really filamentous bulking problems.
Bulking may be one of the main reasons why approximately 50% of U.S.
activated sludge plants don't consistently meet their effluent discharge standards.
It is now known that approximately 25 different filamentous bacteria commonly
occur in activated sludge and each may lead to operational problems. D.H.
Eikelboom in Holland (Water Research 9:365, 1975) provided a rational basis to
"identify" the different filamentous bacteria found in activated sludge. This
identification system is based on filament characteristics as viewed under phase
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contrast microscopy for live samples (in situ) and two simple staining reactions:
the Gram and Neisser stain. Each filament can be "classified" using a four-digit
code, avoiding the earlier problems of lack of specific scientific names. This is
important as many of the filaments found in activated sludge have not been
isolated in pure culture and hence their identity remains unknown. As these
filaments are isolated and properly named (a current research thrust), generic
names replace the four digit number code. Hence, the current list of filaments is a
hybrid between numbers and genus names. Currently there are 24 recognized
filaments (or groups of related filaments in some cases) that cause activated
sludge bulking or foaming. These are given in Table 2.4.
Table 2.4: Recognized Filaments That Cause Activated Sludge Bulking or Foaming (Michael Richard 2003)
-----------------------------------------------------------------------------------------------------------
Sphaerotilus natans Microthrix parvicella*
type 1701 Nocardia spp.**
Haliscomenobacter hydrossis Nostocoida limcola I, II & III
type 021N type 0961
Thiothrix I and II type 0581
Beggiatoa spp. type 0092
type 0914 type 0411
type 0041 type 1863**
type 0675 fungi
type 1851 actinomycetes
type 0803
----------------------------------------------------------------------------------------------------------- * causes both bulking and foaming; ** cause foaming only.
There are six environments or growth conditions that cause the overgrowth of
filaments in activated sludge. Four of these occur in municipal wastewater
systems while all six occur in industrial wastewater systems, with two specific
only to industrial systems (low nutrients and low pH). Many of the filaments have
been associated with other causes in the past, but recent work has indicated the
causes given in Table 2.5 as the primary reason for their growth.
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Table 2.5: Causes of Filament Growth in Activated Sludge (Michael Richard 2003)
------------------------------------------------------------------------------------------------------------
Cause Filaments
1. Low Dissolved Oxygen Sphaerotilus natans
Concentration type 1701
Haliscomenobacter hydrossis
2. Low F/M type 0041
type 0675
type 1851
type 0803
3. Septicity type 021N
Thiothrix I and II
Nostocoida limicola I,II,III
type 0914
type 0411
type 0961
type 0581
type 0092
4. Grease and Oil Nocardia spp.
Microthrix parvicella
type 1863
5. Nutrient Deficiency
Nitrogen: type 021N
Thiothrix I and II
Phosphorus: Nostocoida limicola III
Haliscomenobacter hydrossis
Sphaerotilus natans
6. Low pH Fungi
-----------------------------------------------------------------------------------------------------------
Note that H. hydrossis was previously listed as a low F/M filament. This filament is
caused by low DO, but grows relatively slowly and only occurs at lower F/M and a
longer sludge age. Lower F/M is not its cause, only where it occurs.
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Today, many activated sludge plants regularly monitor the occurrence and
abundance of filaments in their sludge, which has become an important process
control tool. This often leads to prevention of a bulking episode before it becomes
serious.
b. Foaming
Foaming has been attributed to filamentous bacteria (Jenkins et al., 1993; Bitton,
1994). In particular, Nocardia species-Gordonia amarae (formerly Nocardia
amarae), the mycolic acid-containing actinomycetes and “Microthrix parvicella”
have been implicated as the causative agents in foam formation (Davenport et al;
2000). Apart from these, Nostocoida limicola and Type 0041 also have the ability
to produce biosurfactants and are common in activated sludge foam.
A number of filaments resembling the actinomycetes have been observed and
termed ‘Nocardia amarae-like organisms’ (NALO) (Stainsby et al., 2002)
distinguished by their distinctive branching (Soddell, 1999).
Stabilization of biological foams is caused by the following features of foam
causing filaments:
-Production of biosurfactants
-The cell wall hydrophobicity (Wagner, 2006) and
- particle bridging preventing liquid drainage and film thinning (Soddell.J and
Seviour.R.J, 1990)
Nocardia and Microthrix parvicella are known indicators of sludge foaming and
rarely type 1863 also. About 40% of plants in U.S suffer from foaming caused by
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Nocardia sp. Foam causing filaments are easily diagnosed by direct microscopy.
A description of activated sludge foams and their causes is given in Table 2.6.
Table 2.6: Description and Causes of Activated Sludge Foams (Michael Richards, 2003)
Foam Description
Cause(s)
Thin, white to grey foam
Low cell residence time or "young" sludge
(startup foam)
White, frothy, billowing foam
Once common due to nonbiodegradable
detergents (now uncommon)
Pumice-like, grey foam (ashing)
Excessive fines recycle from other
processes (e.g. anaerobic digesters)
Thick sludge blanket on the final clarifier(s)
Denitrification
Thick, pasty or slimy, greyish foam
(industrial systems only)
Nutrient-deficient foam; foam consists
of polysaccharide material released
from the floc
Thick, brown, stable foam enriched in
filaments
Filament-induced foaming, caused by
Nocardia, Microthrix or type 1863
Nocardial foams occur in all types of plants, with no particular association with
specific modes of operation or aeration. Typical nocardial foam occurs as a thick,
stable, "scum" inches to many feet thick on aeration basin and final clarifier
surfaces consisting of activated sludge solids (flocs) and large amounts of
"interlocking" Nocardia filaments. A true Nocardial foam will contain 10-100
fold more Nocardia than the underlying MLSS. Nocardial foam is also known to
contain substantial lipid concentrations, whether is due to the filaments
themselves or the entrapped grease and fat is still unknown. In addition, these
foams contain significant entrapped air, with a bulk density of approximately 7
g/cc. Industrial wastes promoting Nocardia growth (and foaming) include dairy,
meat and slaughterhouse, food processing, pharmaceutical, and any others that
contain a significant amount of grease, oil or fat. Nocardial foaming is also
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associated with high-density restaurant operation in recreational areas (e.g. ski
resorts and summer camps).
Severe Nocardial foam causes number of operational problems and safety
hazards. Foam might also escape to the effluent, increasing its total suspended
solids. In covered aeration basins, foam can accumulate to exceed the available
hydraulic head for gravity flow of wastewater through the basin. Process control
can be compromised if a significant fraction of a plant's solids inventory is present
in the non-circulating foam (e.g. up to 40% of the total solids inventory can be
present in such foams and process control calculations may not be correct).
The most common Nocardia species found in such foams, such as N. amarae, are
not pathogenic; however, other less frequently isolated actinomycete strains are
known opportunistic human pathogens (e.g. N. caviae, N. brasiliensis, N.
asteroides and strains of Mycobacterium). No actual infection has been
documented, however, treatment plant workers and nearby residents may be at
risk.
2.2. Dubai’s Sewage Treatment Plant (DSTP):
The sewage treatment plant located at a place called Al Aweer is the only wastewater
treatment plant in Dubai with the capacity of treating about 260,000 m3 sewage /day
serving the population of approximately 1,400,000 in the city of Dubai , UAE. Both
domestic wastewater and septage are collected by sewers and pumped to the treatment
plant site located about 25 kilometers away from the main city area. The Dubai sewage
treatment plant (DSTP) consists of various treatment stages (Figure 2.3) like preliminary
mechanical, first biological stage comprising of activated sludge process which consists
of six aeration tanks and eight secondary settling tanks. This follows the second
46
biological treatment by fifteen biological trickling filters and eight tertiary settling tanks.
The treated effluent after second biological stages undergoes further treatment by sand
filtration and chlorine disinfection. The large quantities of sludge produced during
various stages of wastewater treatment process is passed through seven consolidation
tanks and finally to five egg shaped sludge digesters where anaerobic digestion of sludge
take place during which sludge is stabilized and part of solids are transferred to methane
gas. The methane gas is used for drying sludge in the final stage of sludge treatment. The
tertiary treated effluent is reused in irrigation, and treated sludge converted to manure for
use as a soil conditioner and fertilizers. Since both wastewater and sludge are reused,
continued successful operation of DSTP is of critical importance to Dubai.
2.2.1 Treatment stages:
� Preliminary Mechanical Stage
Screening and girt removal, pre-aeration to remove H2S
� Mechanical Stage
Settable matter and part of suspended solids removed, 40% BOD removal
� First Biological Stage
Activated sludge process in 6 aeration tanks, microbial action on organic
matter, 90% BOD removal. Sludge flocks produced are removed in 8
secondary settling tanks.
� Second Biological Stage (Biological Filter Stage)
Remnant organic and inorganic pollutants (mainly ammonia) removed in
15 biological filters.The sludge is collected in 8 tertiary settling tanks.
� Sand filtration and Chlorine disinfection
47
Residual suspended solids removal in 8 sand filters, chlorination in two
stages-prior to storage and before effluent discharge.
� Sludge Treatment in three units
First: reduction of sludge volume by removing clear supernatant from
thick sludge
Second: Anaerobic digestion and production of methane gas.
Third: Sludge dewatering in 5 centrifuges and thermal drying, curing for
distribution as fertilizer.
48
RAW WASTEWATER
PRETREATMENT
PRIMARY TREATMENT
SECONDARY TREATMENT
SEDIMENTATION
TERTIARY TREATMENT
FINAL EFFLUENT
[Activated Sludge]
[Screening and Girt removal]
[Flotation and Sedimentation]
[In Secondary Settling Tanks]
[Trickling Filters]
Sand Filtration and Chlorination
SLUDGE
ANAEROBIC DIGESTER
DISPOSAL
Figure 2.3: Generalized Process flow diagram of DSTP
49
2.2.2 DSTP Fact Sheet:
Area: 483,750 Sq Mt.
Capacity: 130,000 m3/day extended to 260,000 m3/day in March 2001
Cost: 400 million AED for first stage and 300 million AED for Second stage.
Total no. of employees: 168
Table 2.7 Chemical analysis of inflowing sewage and outgoing effluent
Parameter Designed Values Operating results (Avg. 2003)
Inflow Outflow Removal% Inflow Outflow Removal%
S.S(mg/L) 325 10 97 245 2.8 99
BOD5(mg/L) 290 10 97 256 2.8 99
COD(mg/L) 680 40 94 569 35 94
NH3
N(mg/L)
38.5 2 95 37 7.9 79
Like any other Sewage treatment plant, the DSTP also frequently faces problems of
bulking and foaming in their secondary settling tanks. The bulking and foaming causes
difficulty in further treatment by biological filters leading to the effluent with unsettled
sludge flocs rendering it unfit for their reuse.
With the current state of knowledge, control of filamentous bulking remains a
challenge facing all engineers, chemists and microbiologists working in the field of
wastewater treatment (Beccari and Ramadori, 1996). Isolation of filamentous organisms
is becoming a promising solution for the investigation of these organisms and factors that
promote or inhibit their growth. Because the majority of filamentous bacteria are
overgrown by more rapid growers, samples are diluted and pretreated before plating on
solid media (Kampfer, 1997). Narrow and short filaments or filaments scarce in the
activated sludge may be concentrated by centrifugation. Another method for selective
isolation of filamentous bacteria is micromanipulation with special micro tools under a
50
microscope (Kampfer, 1997). Although the latter method is one of the latest used
methods for isolation of filamentous bacteria, it requires highly specialized equipment, is
very expensive and therefore inaccessible to many laboratories. It also tends to be very
labour intensive and difficult (Ramothokang et al., 2003). It is important that the flocs be
broken up before isolation so as to facilitate easier separation of filamentous bacteria
from floc-forming bacteria. This is so as to avoid or minimize competition by faster
growing floc-formers on different solid media. The filamentous micro-organisms
traditionally have been identified by their morphology and simple staining reactions as
described by Eikelboom & van Buijsen (1983) and Jenkins et al. (1986). The majority of
filamentous bacteria in sludges, however, are still unidentified beyond these simple
characteristics (Seviour & Blackall, 1999). Over the last decade, molecular biological
methods have been used to identify and monitor filamentous micro-organisms (Blackall,
1994; Wagner et al., 1994; Bradford et al., 1996; Erhart et al., 1997; Kanagawa et al.,
2000; Bjornsson et al, 2002). In particular, fluorescence in situ hybridization (FISH)
using 16S rRNA-targeting oligonucleotide probes (DeLong et al., 1989) is an invaluable
technique for directly identifying micro-organisms in their natural settings (Amann et al.,
2001).
2.3 In-situ Detection Technique:
Use of radioactively labeled rRNA-directed oligonucleotide probes (In situ hybridization)
for the microscopic detection of bacteria was introduced by Giovannoni et al. (1988).
Soon after, radioactive labels were replaced with non-isotopic dyes, which were both
safer and simpler to use. DeLong et al. (1989) were the first to use fluorescently labelled
oligonucleotides as probes for the detection of individual microbial cells. Detection and
characterization of bacteria has been faster and easier with the development of such
51
molecular biological methods which do not require the isolation and enrichment of
bacterial strains. Fluorescent in situ hybridization (FISH) is one of the most commonly
used molecular methods for the identification of microorganisms in Wastewater
Treatment Plants (WWTP).
2.3.1 Ribosomal RNA based detection
Use of ribosomal RNA (rRNA) genes as molecular markers in molecular methods such
as FISH is advantageous because of the following reasons.
All living cells contain ribosomes, which are part of the cell’s apparatus for translating
deoxyribonucleic acid (DNA) into protein. rRNA is a dominant cellular macromolecule.
Most bacterial cells have about 103 - 10
5 ribosomes. This natural amplification results in
excellent sensitivities of hybridization assays (Amman, 1995).
The RNA content within cells varies depending on the general metabolic activity or
growth rate of a given species.
RNA molecules contain conserved and variable regions which make it possible to find
general as well as specific target sites for probes. These regions are used for identification
purposes. Hence rRNA are excellent molecules for discerning evolutionary relationships
among bacteria.
A practical reason for using rRNA is the public availability of large databases. They
have enough sequence information to be used as a phylogenetic marker (Maidak et.al
1999).
Thus the rRNA approach has become a widely used method for studying the microbial
community structure of natural and man-made environments in a truly quicker and
cultivation-independent way.
52
2.3.2 Hybridization
The artificial construction of a double-stranded nucleic acid by complementary base
pairing of two single stranded nucleic acids is called hybridization.
Group and genus specific fluorescently labelled rRNA-targeted oligonucleotide probes
(short sequences of nucleic acids which are complimentary to a specific sequence of
RNA) were used to analyze directly the community structure of organisms in biological
WWTP, particularly in activated sludge systems by in situ hybridization. There are two
different hybridization assays which have been commonly used in microbial ecology
studies: slot-blot hybridization and fluorescent in situ hybridization (FISH). Slot-blot
hybridization requires the extraction of nucleic acids from samples to be tested.
Subsequently, nucleic acids are immobilized on membranes and hybridized with
radioactive or non-radioactive probes. With FISH, the target nucleic acids are detected
directly in the cells. To achieve in situ detection, cells should be permeabilized to allow
the probe access to the inside of cells. At the same time, the morphological integrity of
the examined cells should be maintained. This is usually achieved by fixing the cells with
alcohols or aldehydes (Amman, 1995). Probes labeled with a fluorescent dye bind to a
signature sequence in the ribosomal RNA of the target organism(s) of interest during the
hybridization procedure (Stahl and Amman, 1991) (Following Fig 2.4).
Figure 2.4: Base pairing between a fluorescently labeled oligonucleotide probe and a
target rRNA
53
Hybridization takes place at an optimal temperature which is a function of the base
composition of the probe and the complementary target sequence. This is determined
empirically to avoid non specific binding of the probe to the rRNA sequences.
Optimization of hybridization conditions is done by including different concentrations of
formamide in the hybridization buffer at a single temperature (Manz et.al 1992).
Addition of formamide is one of the simple ways to discourage hydrogen bonding. It
facilitates denaturation of the probe and the target DNA. During the hybridization step,
the relatively high formamide concentrations favor probe-target annealing. In other
words, relatively high formamide conditions increase the hybridization stringency
(Gerhardt et.al 1994). Binding of the fluorescently labeled probe to the target rRNA
sequences allows visualization and enumeration of individual cells with the help of
epifluorescence microscope.
However, there are a number of problems with the technique:
1) Permeabilization: Successful entry of the probe into the cell is the first and important
step of the FISH technique. Most microorganisms have been permeable to short
oligonucleotide probes following fixation (Giovannoni et.al 1988 and Delong et.al 1989).
Although a variety of fixatives have been evaluated, autofluorescence is generally
minimized by fixation in formaldehyde.
2) Uneven cell penetration: Successful cell permeabilization does not guarantee
hybridization of the targeted rRNA sequence with a probe (O’Donnell 1997). It is
uncertain whether oligonucleotide probes will be able to permeate all cell types and find
16S rRNA target sequences (Muyzer et.al 1995). Hence high stringency is required to
manage even cell penetration.
54
3) At times no fluorescence signal is obtained because of a very low concentration of
cells or low RNA content. It is found that cells less than 103-10
4 per ml can cause poor
signal detection (Amman et.al 1995). The sensitivity of the technique increases with an
increased number of metabolically active cells.
4) The target sequence in the rRNA is believed to be inaccessible due to strong
interactions with ribosomal proteins or highly stable secondary structure elements of the
rRNA itself. If the pure culture cells give a strong hybridization signal with a universal
probe while they are not giving signal with specific probe, this generally indicates poor
accessibility of the target site. In situ accessibility can sometimes be improved by the
addition of formamide to the hybridization buffer.
5) High amount of background auto fluorescence: The auto fluorescence of some
bacteria, such as phototrophs (Muyzer et.al 1995), and the background fluorescence of
inorganic particles is often much stronger than the fluorescence of the specific probe
binding. Bleaching of fixed cells before hybridization and use of fluorescent dyes with
emission wavelengths that do not coincide with the auto fluorescence can minimize the
background fluorescence (Delong et.al 1989).
2.3.3 Advantages of FISH
FISH permits rapid, simple and accurate detection of related groups of bacteria. FISH
allows in situ detection of bacterial species without requiring culturing the bacteria. As a
result, it is a potential tool for the identification of microbial communities in wastewater
treatment plants. This technique also allows the characterization of bacterial species
which have not been cultured yet. Therefore, undiscovered diversity may be
characterized by FISH. Another advantage of FISH is that it can provide phylogenetic
information based on 16S rRNA sequences and therefore it can be utilized to distinguish
55
between different populations independent of activity. The probes can also be designed
for different levels of specificity. More conserved regions allow differentiation between
large phylogenetic entities like the Archea, Bacteria and Eukaryota domains, and they
also serve as targets for universal probes that react with all living organisms. Variable
region sites can be used to identify certain genera, species, and infrequently also for
subspecies or even for a certain strain (Stahl and Amman, 1991).
Observation of the location of different bacterial species and their abundance at various
places in activated sludge flocs may improve our understanding of the complex
microbiological processes. In theory, in situ growth rates and physiological activities may
be estimated by measuring the fluorescence conferred by the rRNA-targeted
oligonucleotides in combination with digital image analysis as the cellular quantity of
rRNA is closely related to the growth rate of cellular micro-organisms. Quantification of
the probe conferred signal intensity of single cells seems to be an appropriate tool for
estimating their physiological state in situ (Delong et.al 1989; Poulsen et.al 1993). It has
been demonstrated that there is a linear relationship between the average fluorescence
intensity per cell volume and the growth rate of the cell culture (Delong et.al 1989;
Poulsen et.al 1993; Kerkhof et.al 1993). Therefore, growth kinetic parameters may be
determined with this technique.
Until recently, the enumeration of microorganisms was limited to cultivation-based
methods. These methods underestimate the number of bacterial cells. FISH allows the
detection of one to three orders of magnitude more cells than plate counts in
environmental samples; 60-90% of all cells present in activated sludge can be detected
with DNA-intercalating dye (DAPI). FISH can also visualize a similar quantity of cells.
56
Enumeration of bacterial species in activated sludge systems using FISH has been
previously investigated successfully (Coßkuner, 2000 and Davenport et.al 2000).
Quantification of microorganisms is central to microbial ecology and environmental
engineering. The definition of the numbers of a key functional group is very important
since changes in functionality will reflect the change of bacterial numbers. The operation,
control and design of the biological WWTP and inhibition studies are directly related to
the numbers of functional groups of microorganisms. FISH seems to be a powerful tool
in future research for the quantification of target groups of organisms in WWTP.
2.3.4 Applications of FISH in Wastewater Treatment Plants
Identification of filamentous and non-filamentous bacteria in WWTP, particularly in
activated sludge processes was extensively studied. Microorganisms related to phosphate
removal, nitrification process and bulking and foaming problems were also investigated.
Enhanced biological phosphate removal (EBPR) in anaerobic-aerobic activated sludge
systems has generally been linked to Acinetobacter spp whose affiliation with the
filamentous bacterium of the Eikelboom type 1863 has been established using FISH
technique.
No definite ways have been found to prevent or control the accumulation of stable, often
chocolate-colored, viscous foam or scum on the surfaces of activated sludge aeration
tanks. In addition, it has been observed that strategies that work in one WWTP, may not
work in another. Therefore, it is necessary to understand both the taxonomic diversity and
ecology of the organisms which cause the foaming problem. The true extent of the
taxonomic diversity and identity of Actinomycete were studied using FISH (Goodfellow
et.al 1996). Nitrification is the necessary first step in the complete removal of nitrogen by
nitrification-denitrification processes since wastewater almost always contains the
57
reduced forms of nitrogen, mainly as ammonia and organic nitrogen. Autotrophic
nitrification is achieved by a two-step biological oxidation process. In the first step,
ammonia is oxidized to nitrite by ammonia oxidizing bacteria (AOB), which are often
represented by the genus Nitrosomonas. In the second step, nitrite oxidation is carried out
by nitrite oxidizing bacteria (NOB) to produce nitrate. In situ hybridization studies using
16S rRNA targeted probes highlighted the importance of non-Nitrobacter NOB for the
nitrification process (Juretschko et.al 1998). Further Schramm et al. (Schramm, 1996)
have studied the in situ localization of Nitrosomonas spp. and Nitrobacter spp. In another
study, localization of AOB at the centre of activated sludge flocs was also established.
FISH enables detection of nucleic acid sequences by a fluorescently labeled probe that
hybridizes specifically to its complementary target sequence within an intact cell (Amann
et al., 1995). Typically, oligonucleotide probes are between 15 and 30 bp in length and
are usually labelled at the 5’-end of the probe.
Most commonly used dyes for FISH are fluorosceinisothiocyanate [FITC] (a fluorescein-
derivative), Texas Red (a rhodamine-derivative) or Cy3 or Cy5 (cyanine dyes). Detection
of two or more microorganisms can be achieved simultaneously by selecting
fluorochromes with different excitation and emission maxima. In addition, blue
fluorescent counterstaining can be performed with aromatic diamidines such as DAPI (4,
6-diamidino-2-phenylindole dihydrochloride) that binds with high affinity to DNA.
Prior to hybridisation, bacteria have to be fixed and permeabilised for penetration of the
fluorescent probes into the cell and to protect the RNA from degradation by endogenous
RNAses. Such fixation should result in maximum retention of target RNA, maintenance
of cell integrity and morphological detail, while allowing for sufficient probe access.
Following fixation, hybridisation is achieved by incubating the sample with pre-heated
58
hybridisation buffer containing the probe. Proper annealing of the probe to the target is
achieved under stringent conditions. Stringency can be adjusted by varying either the
concentration of formamide in the hybridisation buffer or the hybridisation temperature.
Formamide decreases DNA melting temperature, allowing for lower temperatures to be
used with high stringency. Hybridisation is followed by washing. Samples undergo post-
hybridisation stringency washes, often regulated by varying the salt concentration in the
washing solution.
In the current application, the target molecule for FISH is 16S rRNA. Each taxonomic
level, down to genus-specific and species-specific, can be detected by designing
oligonucleotide probes according to the region of rRNA targeted, reviewed
comprehensively by Amann et al. (1995).