23

CHAPTER 2

Review of Literature

24

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.

25

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).

26

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

27

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:

28

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.

29

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

30

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)

31

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.

32

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).

33

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

34

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)

35

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

36

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.

37

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

38

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

39

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

40

(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

41

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.

42

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.

43

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

44

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

45

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).