STUDIES OF BIODEGRADATION OF NITROPHENOLS

ABSTRACT THESIS

SUBMITTED fOR THE AWARD OF THE DEGREE W

Mottot of $I)tIos(09i)p IN

CHEMISTRY

BY

FARAH KHAN

Under the Supervision of

Dr. SUHAIL SABIR

DEPARTMENT OF CHEMISTRY ALIGARH MUSUM UNIVERSITY

AUGARH (INDIA)

2010

Abstract

The thesis entitled "Studies of Biodegradation of Nitrophenols" deals with aerobic

granules of few organic xenobiotics with an aim to determine various parameter of

aerobic granulation such as degradation rate, removal efficiency, biomass

concentration (MLSS and MLVSS), Optical density (O.D. oo) and degradation profile

of COD and the compound. This work is based on aerobic granulation technology.

The aerobic granulation technology is a recent cell immobilization technique like

biofilm formation but here no carrier material is present. It was proved earlier that

microbes in suspension form are more efficient in degrading organic compounds.

Aerobic granulation is only reported in Sequencing Batch Reactor (SBR). Due to

some predetermined factors like H/D ratio, superficial air velocity and settling time,

microbes form aggregates which become more and more compact with the passage of

time. Such compact granules settle faster leading to high active biomass retention and

have high resistance to toxic compounds. Initially granules were cultivated using less

toxic substrates like glucose and acetate. Now this technology is used for cultivafing

aerobic granules using highly toxic compounds as well. This technology highlights

the effectiveness of granules over organic chemicals as food for reactors and also

useful for treating higher load and fluctuations.

The experiment was conducted in a laboratory scale SBR reactor with a working

volume of 1.4 1. Reactor was operated sequentially with fill, react, settle, draw and

idle period for a cycle. Nutrient (macro and micro) and toxic organic compounds were

also supplemented during acclimation period and in SBR cycle.

Chapter 1 deals with Introduction- An ovevYicM' on Biodegradation of Organic Toxic

Compounds through Aerobic Granulation Technology in SBR. It describes an

overview on Water pollution. Wastewater treatment. Water quality parameter and

TerniinologA used.

Chapter 2 thesis deals with "Biodegradation of Phenol by Aerobic Granulation

Teclmolog}' in Sequencing Batch Reactor." This chapter describes properties, uses

and health hazard information of phenol. The Phenol is fed into a SBR of height and

1

diameter ratio (H/D 30) and operated at-4 liours cycle with a volumetric exchange

ratio of 50% resulting in a constant HRT (Hydraulic retention time) of 8 hours.

Degradation of phenol was completed in 60 days. Maximum phenol concentration of

650 mg r' is treated efficiently which is equal to an organic load of 3.9 kg m" d"'.

Mixed liquor suspended solid (MLSS) and mixed liquor volatile suspended solid

(MLVSS) were stabilized at 7.3 and 4.72 gm 1"'. Effluent and SBR O.D.goo stabilizes

at 0.15 and 2.64 which corresponds to minimum loss of biomass. Phenol removal

efficiency and COD removal efficiency of 94.3% and 97.39%) were achieved. After

granulation the effluent phenol concentration and COD were 37 mg f' and 193 mg f'

respectively. Degradation studies show that initially the removal rate of phenol was

high but later on it decreases. Low concentration of 50 mg 1"', 100 mg f' and 200 mg

r' phenol comes down below detection limit in about first 150 minutes of the SBR

cycle but high concentration of 400 mg 1"' and 650 mg 1"' took 240 minutes for above

90%) removal. In a single cycle, degradation rate of phenol was 153.24 mg f' hr"' at

650 mgf'.

Chapter 3 deals with ''Aerobic Granulation for Para nifro phenol Biodegradation in

a Sequencing Batch Reactor. " This chapter describes properties uses and health

hazard information of para nitro phenol (PNP) along with the studies on degradation

of PNP in SBR. This SBR cycle covers 90 days degradation. The effect of high

organic loading rate 2.1 kg m"'' d"' at 350 mg f' concentration of PNP on granules was

investigated. The SBR cycle was kept lor 4 hours with 8 hours HRT (50%> volumetric

exchange ratio). Granules were compact and strong and were capable to degrade PNP

completely. The COD and PNP removal efficiencies were found 95.32%o and 96%).

MLSS, MLVSS, O.D.600 of effluent and SBR were stabilized at 5.9 gm 1"', 3.5 gm 1"',

0.35 and 2.53 respectively. The degradation profile of COD and PNP in a single cycle

reveals that initially the removal rate was fast but ultimately decreases later. Low

concentrations of 40 ma, !"'. 80 mg f' and 150 m<z f' were desjraded in the first 170-

180 minutes but degradation of higher concentration of PNP i.e. 250 mg f' and 350

mg r' occurred in 230-240 minutes. Degradation rate of PNP at 40 mg 1"' and 350 mg

r' concentration were 9.45 mg f' hr'' and 83.97 mg f' hr'' respectively.

Chapter-4 deals with "Biodegradation of 2,4-dinUrophenol by Aerobic Aggregates in

Sequencing Batch Reactor." In this chapter the properties uses and health hazard

information of 2,4-dinitrophenol (DNP) are described. In sequencing batch reactor

(SBR) DNP was fed and investigated. SBR was operated on 6 hours cycle at 50%

volume exchange ratio resulting HRT of 12 hours. Whole SBR cycle was completed

in 90 days (30 days for acclimatization and 60 days for SBR operation) and 0.48 kg

m^ 1"' load of DNP was degraded. SBR O.D.goo, effluent O.D.eoo, stabilized at 2.63,

0.29 respectively. MLSS and MLVSS were found to be 6.1 and 3.98 respectively.

DNP and COD removal efficiency were 88% and 97.21% respectively. The

degradation profiles of COD and DNP shows a high initial rate which decreases later.

A high removal rate was observed in the first hour of cycle and after the rate

decreases. At highest concentration degradation rate and removal efficiency of DNP

were 17.58 mg f' hr"' and 88% respectively. At highest concentration COD removal

efficiency was 97.21%) respectively.

This study demonstrated the successful cultivation of aerobic granules in SBR using

phenol, PNP and DNP. These aerobic granules can handle high strength industrial

wastewater containing phenol, PNP and DNP which otherwise causes the failure of

conventional activated process. In this technique bacterial coaggregation might be an

integral component.

STUDIES OF BIODEGRADATION OF NITROPHENOLS

THESIS

SUBMITTED rOR THE AWARD OF THE DEGREE OF

Mottov of $^tIo£fopI)|' IN

CHEMISTRY

BY

FARAH KHAN

Under the Supervision of

Dr. SUHAIL SABIR

DEPARTMENT OF CHEMISTRY ALIGARH MUSUM UNIVERSITY

AUGARH (INDIA)

2010

z ' v t

T8370

to My

(Dearest (parents

Or. SutiaJi Sabir

OEFARTMEI^T OF CHEMISTRY Aho^arh S-lusl'in ( tiivcrsilv.

AhouHi - 2(Cnii2

CERTIFICATE MU z^^^o^^

This is to certify that the work embodied in this thesis entitled "Studies of

Biodegradation of Nitrophenols" is the result of the original work of Ms Farah Khan

carried out under my direct supervision and is suitiible for submission for the award of

the degree of Ph.D. in Chemistry of Aligarh MusHm University.

(Dr. SuhaU Sabir)

Residence: 26 Nishat Aparis Shamshad Market, Ahaarh- 202002 (INDIA) e-riai! sutmiitabii'^ernail com

STATEMENT

/ hereby declare thai the matter embodied in this thesis is the result of investigation

carried out by me in the Department of Chemistry AMU Aligarh, India, under the

supervision of Dr. Suliail Sabir.

In keeping with the general practice of reporting scientific observations, due

acknowledgement has been made wherever the work described is related on the

findings of other investigators.

^ ^ ^

Farah Khan

V4\A,A.^

ACKNOWLEDGEMENTS

First of all my heartfelt gratitude to almighty Allah for everything 1 am today. He

guided me through every thick and thin through innumerable and invisible ways to

fulfill this endover of mine.

I would express my deepest thanks and respect to my supervisor Dr. Suhail Sabirfor

showing me the confidence and support that a student ever need. Without his able and

experienced guidance surely this work would have been incomplete. He could not

even realize how much I have learned from him.

Special thanks to the chairman, Department of Chemistry AMU, Aligarh, for

providing me the necessary research facilities and material for conducting my

experiments.

I would like to give my heartfelt thanks to Mohd. Zain Khan for supporting and

helping me throughout my research work. He has given me his precious advice and

generous help to bring out this thesis. I wouldn 't have been able to complete my work

as efficiently without his constant cooperation.

My sincere thanks to Shams Qamar Usmani for helping me planning and starting my

work, who was always cooperative and cordial, whenever I needed his guidance in

my research work.

My lab mates have been a source of constant inspiration for me. They helped me

always, sometime with criticism and sometimes with their hearty laugh to ease my

tensions. 1 thank Dr. Rashid, Dr. Ishaat, Dr. Niyazi, Dr. Tanveer, Pradeep Kumar,

Mohd. Mujahid, Fakhra, Dr. Salma Tabbssum, Sadaf Haneef Noorussaba, Neeti,

Saima Sultana and Dr. Zoya Zaheer.

My loving thanks to all my friends Shahzia Saleem, Dr. Sana Siddiqui, Tazeem

Fatima, Sadaf Afrin, Nazia Malik, Divya, Dr. Imran Khan, Dr. Taiisif Altamash, Dr.

Mudassir, Meraj Alam who filled my world with the charms of their unique

personalities. My special thanks to AH Adeel Qamar, Dr. Sudhanshu Sharma, Dr.

Iram Siddqui, Huma Haider, Nida Qutub who with their intelligent and innovative

ideas and encouragement have helped me in compiling my thesis.

I would express my humble gratitude towards my family whose unending support

helped me to fulfill this ambition. Their unconditional love, support and blessing

made me to chase my dreams. I feel honored to thank my father Mr. Mohd. Abbad

and my mother Mrs. Akhtar Jamal who stood by me through every painful and happy

moment and made me believe that my world is empty without them. Their constant

and unconditional faith in me has really contributed in the culmination of this work.

My special thanks to my sister Naheed Akhtar, brother-in-law Abdul Lateef my

brother Javed Akhtar, sister-in-law Samra Khan, my loving nieces Rimsha Khan,

Sarah Khan, Bushra Khan and mama Rajan Mishra who always gave me a reason to

look positive towards every development in my life.

' ^ '

Farah Khan

CONTENTS

Page No.

CHAPTER 1

1.1

1.2

1.3

CHAPTER 2

2.1

2.2

List of Figures

List of Tables

I

V

Introduction- An overview on 1-45

Biodegradation of Organic Toxic

Compounds through Aerobic Granulation

Technology in SBR

Water Pollution 2-5

Wastewater Treatment

Water Quality Parameters

Terminology

References

6-29

30-35

36-38

39-45

Biodegradation of Phenol by Aerobic 46-91

Granulation Technology in Sequencing

batch reactor

Phenol 46-53

Biodegradation of Phenol by Aerobic 54-80

Granulation Technolocx'

References 81-91

CHAPTER 3

3.1

Aerobic Granulation for Para nitro phenol 92-133

Biodegradation in a Sequencing Batch

Reactor

Para Nitro Phenol 92-98

3.2 Biodegradation of Para Nitro Phenol by 99-122

Aerobic Granular Sludge Process

References 123-133

CHAPTER 4 Biodegradation of 2,4-dinitrophenol by 134-171

Aerobic Aggregates in Sequencing batch

reactor

4.1 2,4-dinitrophenol 134-140

4.2 Biodegradation of 2,4-dinitrophenol in 141-161

Sequencing Batch Reactor

References 162-171

List of Publications VII

LIST OF FIGURES:

FIGURE NO. TITLE p^^^, ^^

Design principle of steps of sequencing Fial.l 18

batch reactor (SBR). 'o

Fig 2.1 Structure of phenol. 48

Fig 2.2 Resonance structure of phenol. 48

Fig 2.3 At the time of acclimatization with 66 'e

phenol in activated sludge process.

SBR while aeration during a cycle (with Fig 2.4 66

phenol).

Fig 2.5 SBR at the time ofsettling (with phenol). 67

Settled sludge after aeration (with Fis 2.6 67

phenol). 'o •

Variation of MLSS and MLVSS

Fig 2.7 concentration of phenol during its 72

degradation.

Microscopic image of sludge after one Fig 2.8 a 72

week of inoculation.

Fig2.8b&c SEM image of mature granules at 100 73

and 15,000 magnifications.

Fig 2.9 Optical Density (600nm) of effluent and 76

SBR variation with time.

Concentration profile of phenol during Fig 2.10 76

single cycle. 'fe

COD removal profile of phenol during Fig 2.11 77

single cycle operation.

Percent COD removal efficiency along Fig 2.12 77

with effluent and influent COD.

Fig 2.13 Percent phenol removal Vs effluent and

influent concentration.

' o •

80

Fig 3.1 Structure of PNP. 94

Fig 3.2 Resonance structure of the PNP anion. 94

Fig 3.3 At the time of acclimatization with PNP 107

in activated sludge process.

SBR while aeration during a cycle (with Fig 3.4 107

PNP).

Fig 3.5 SBR at the time of settling (with PNP). 108

Fig 3.6 Settled sludge after aeration (with PNP). 108

Variation of MLSS and MLVSS Fig 3.7 _ 114

concentration during PNP degradation.

Microscopic image of granules after one Fig 3.8 a ; 114

week, of inoculation.

II

SEM image of mature granules at 4,000 Fig3.8b&c 115

and 15,000 magnifications.

Optical Density (O.D.m) of SBR and

Fig 3.9 effluent during the operation. 117

Concentration profile of PNP during FigS.lO . 117

single cycle operation.

COD removal profile at different PNP

Fig 3.11 concentration during single cycle 119

operation.

COD variation alon^ with % removal Fig 3.12 ^ 119

efficiency during the study. 'to

Variation of PNP along with % removal Fig 3.13 . 121

efficiency during SBR operation.

Fig 4.1 Structure of DNP. 13

Fig 4.2 Resonance structure of the DNP anion. 136

Fig 4.3 At the time of acclimatization with DNP 147

in activated sludge process.

SBR while aeration during a cycle (with Fig 4.4 147

DNP).

Fig 4.5 SBR at the time ofsettling (with DNP). - 148

III

Fig 4.6 Settled sludge after aeration (with DNP). 148

Variation of MLSS and MLVSS

Fig 4.7 concentration of DNP during its 154

degradation.

Microscopic image of sludge after one Fig 4.8 a 154

week of inoculation.

SEM image of mature granules at 4,000 Fig4.8b&c 155

and 15,000 magnifications.

Variation of Optical Density (O.D.600) of Fig 4.9 " 157

SBR system with time.

Concentration profile of DNP during Fig 4.10 157

single cycle operation.

COD removal profile DNP during Fig4.11 " 159

degradation in single SBR cycle.

COD variation along with % removal Fig 4.12 159

efficiency during the study. '&

Variation of DNP along with % removal Fig 4.13 _ 160

efficiency during SBR operation.

IV

LIST OF TABLES:

TABLE NO. TITLE PAGE NO

Table 1.1 Classification of Water 4

Pollutants.

Table 1.2 Wastewater Treatment 12

Processes.

Table 1.3 Standards of Water Quality 31

Parameters.

Table 1.4 Standard Concentrations of 34

Drinking Water Components.

Table 2.1 Characteristics of Major 55

Phenolic Wastewater.

Table 2.2 Organic Loading Rate of 57

Phenols in Different Mediums.

Table 2.3 Amounts of Nutrients (phenol). 69

Table 3.1 Organic Loading Rate of PNP in 101

Different Mediums.

Table 3.2 Amount of Macronutrients 110

(PNP).

Table 3.3 Amount of Micronutrients 110

(PNP).

Table 4.1 Organic Loading Rate of DNP in 143

Different Mediums,

V

Table 4.2 Amount of Macronutrients 150

(DNP).

Table 4.3 Amount of Micronutrients 150

(DNP).

VI

CHAPTER 1

INTRODUCTION- AN O V E R V I E W O N

BIODESRADATION OF ORGANIC TOXIC

COMPOUNDS THROUGH AEROBIC

GRANULATION TECHNOLOGV IN SBR

Introduction-An Overview on Biodegradation of Organic Toxic

Compounds through Aerobic Granulation Technology in SBR

Pollution is the contamination of harmful constituents to water, soil and air which

ad\'ersely alter the natural quality of the environment. Environmental pollution is

actually the result of urban industrial technological revolution and speedy exploitation

of every bit of natural resources. The discharging of waste into the environment was

the way to eliminate them. The permitted discharge levels have been vastly exceeded,

causing such environmental contamination that our natural resources cannot be used

for certain purposes and their characteristics have been altered. Dyes, phenols,

pesticides, fertilizers, detergents and other chemical products are disposed of directly

into the environment, without being treated.

Depending on the type of pollutant, pollution can be named as air pollution, soil

pollution noise pollution, thermal pollution, pesticide pollution, radiation pollution

and water pollution. Gaseous, liquid and solid substances which tend to interfere with

human comfort, health or welfare and cause environmental damage, causes air

pollution. Various fertilizers and other chemicals, which are added to increase the

crop yield and to prevent the crop from pests, adversely affect the soil properties and

causes soil pollution. Noi.sx' pollution is occurred when sound of sufficiently high

intensity causes ill effects on our ears. Thermal pollution is the discharge of waste

heat via energy dissipation into cooling water and subsequently into nearby

waterways. Pesticides are organic and inorganic chemicals, their effectiveness has

caused considerable peslicick pollution. Radiation pollution is any form of ionizing or

nonionizing radiation that results from human acti\ities.

A large number of organic substances are nowadays introduced into the water

system from various sources such as industrial effluents, agricultural runoff and

chemical spills. Their toxicity, stability to natural decomposition and persistence in

the emironment has been the cause of much concern to societies and regulation

authorities around the world. Phenols and nitrophenols from the industrial wastewater

1

has become an issue of interest during the iast few years because of the toxicity of

these chemicals. The research presented in this thesis describes the development of

wastewater treatment through aerobic granulation technology in sequencing batch

reactor.

1.1 WATER POLLUTION;

Water pollution is a major problem in the global context. Water pollution is the

contamination of water by foreign matter which deteriorates the quality of water or it

is a state of deviation from the pure condition, whereby its normal functions and

properties are affected. Water pollution covers pollutions in liquid forms, liquid

pollution occurs in the oceans, lakes, streams and rivers. Water is typically referred

as polluted when it is impaired by anthropogenic contaminants this neither support a

human use nor undergoes a marked shift in its ability to support its constituent,

bioticse.

1,1.1 Causes of Water Pollution:

The specific contaminants leading to pollution in water includes a wide spectrum of

chemicals, pathogens and physical or sensory changes such as elevated temperature

and discolouration. While many of the chemicals and substances that are regulated

may be naturally occurring (calcium, sodium, iron, manganese, etc.) the concentration

is often the key in determining the natural component of water and contaminant.

Other natural and anthropogenic substances niay cause turbidity (cloudiness) which

blocks light and disrupts plant growth, and clogs the gills of some fish species.

The signs of water pollution are obvious to all;

• Bad taste of drinking water.

• Offensive odour from lakes. ri\'ers and ocean beaches.

• Unchecked growth of aquatic weeds in water bodies.

• Decrease in number offish in fresh water, river water and sea water.

• Oil and grease floating on water surface.

1.1.2 Types of Water Pollutant:

Sodhi, (1988) classified water pollutants in to four categories: Chemical, physical,

physiological and biological (Table 1.1). The introduction of these contaminants into

the aquatic systems is likely to cause health hazards, harm to ecology, damage to

structures or amenities and interference with legitimate uses of water.

1.1.3 Effects of Water Pollution:

The effects of water pollution due to agricultural, fertilizers, chemicals and

contaminated ground water are given below.

• Contaminated Ground Water Effects

If contaminated water enters the ground, there may be serious effects. There is a

probabilit}' of developing liver or kidney problems and cancer or other illnesses. In

rivers, oceans and seas, water pollution effects flora and fauna in them. Further, the

birds and animals that consume this contaminated food supply can be perished.

• Fertilizers and Other Chemicals Effects:

Nitrates in drinking water leads to diseases to infants that may lead to their death.

Cadmium is a metal in sludge-derived fertilizer, can be absorbed by crops. When

people ingest this, they may suffer from diarrhoeal disorders, liver and kidney

damage. The inorganic substances like mercury, arsenic and lead most poisonous and

cause pollution. Other chemicals can also lead to problems concerning the taste, smell

and colour of water. Pesticides, PCBs and PCPs are all poisonous to all sorts of life.

Table 1.1: Classification of Water Pollutants:

CHEMICAL PHYSICAL PHYSIOLOGICAL BIOLOGICAL

Organic pollutant Colour

(Soap, detergents,

dyes etc.)

Inorganic pollutant Turbidit}'

(Acids, alkaline,

cations, anions)

Suspended matter

Forth

Radioactivity

Thermal

Taste

Odour

Weeds

Alcae

Viruses

Bacteria

Protozoa

Parasitic worms

• Effects of Agricultural Water Pollution:

Rain and irrigation water drains off cultivated land that has been fertilized and treated

with pesticides, the excess nitrogen and poisons are mixed with it into the water

supply. These pesticides are toxic and pollute the water in a different mode. Aquatic

plants growth cause de-oxygenation of water and annihilate flora and fauna in a

stream, lake and river. Fertilizers enhance the growth of bacteria that are preventing

in water to hazardous levels.

• Some Other Effects:

Blood diseases, nervous system disorders and heart diseases are some of the common

effects of water pollution. Many toxins in polluted water lead to cancer. Often, the

body's chromosomal makeup can also be altered. Some of the less potent effects are

skin lesions, vomiting and diarrhoea.

To avoid these harmful effects of water pollution, wastewater treatment is necessary

using different methods mentioned in wastewater treatment process (1.2);

1.2 WASTEWATER TREATMENT PROCESSES:

Wastewater contains organic and inorganic components in a complex mixture of

compounds botli dissolved and solids. In a treatment plants the contaminants must be

eliminated to acceptably low concentration.

Disposal of wastewaters from an industrial plant is a difficult and costly problem.

Most petroleum refineries, chemical and petrochemical plants (Beychok et al., 1967

and Tchobanoglous et al., 2003) have onsite facilities to treat their wastewaters. Other

industrial processes that produce a lot of wastewaters such as paper and pulp

production have created environmental concern leading to development of processes

to recycle water, used within plants before they have to be cleaned and disposed off

(Byrdetal., 1984).

There are numerous processes that can be used to clean up wastewaters depending on

the type and extent of contamination. Physical, chemical and biological treatment

processes are used to treat wastewater (Table 1.2). Most wastewater is treated in

industrial-scale wastewater treatment plants (WWTPs). Physics, chemistry,

microbiology and engineering are all involved in purifying wastewater so that it can

be safely returned to the environment.

While the devices used in wastewater treatment are numerous and will probably

combine physical, chemical and biological methods. The toxic, nonbiodegradable

chemicals in industrial waste effluent can be purified by generally grouped under

these methods:

I. Preliminary Treatment:

The principal objectives of this treatment are the removal of gross solids i.e. large

Heating and suspended solid matter, grit, oil and grease if they are present in

considerable quantities. At most plants, preliminary treatment is used to protect

pumping equipment and facilitate subsequent treatment processes.

11. Primary Treatment:

The purpose of primary treatment is to reduce the velocity of the wastewater

sufficiently to permit solids to settle and floatable material to surface and also to

facilitate secondary treatment. After the removal of gross solids, gritty material and

excessive quantities of oil and grease, the next step is to remove the remaining

suspended solids as much as possible.

III. Secondary Treatment:

In this treatment, the dissolved and colloidal organic matter present in wastewater is

removed by biological processes involving bacteria and other organisms. These

processes are anaerobic or aerobic.

IV. Tertiary Treatment:

This final treatment has come into use to describe additional treatment following

secondary treatment. Tertiary treatment has been used to describe processes which

remove plant nutrients, primarily nitrogen and phosphorous from

wastewater. Improvement and upgrading of wastewater treatment units as well as the

need to minimize environmental effects has led to the increased use of tertiary

treatment.

A complete treatment system may consist of the application of a number of physical,

chemical and biological processes to the wastewater.

1.2.1 Physical Process:

This process usually treats suspended rather than dissolved pollutants. It may be a

passive process, such as simply allowing suspended pollutants to settle down or float

to the top naturally depending on whether they are more or less dense than water. The

process ma}' be aided mechanically, such as b\ gently stirring the water to cause more

particles to bump into each other and stick together, forming larger particles which

will settle or rise faster, a process known as, flocculation. Chemical flocculants may

also be added to produce larger particles. To aid floatation process dissolved air under

pressure may be added to cause the formation of tiny bubbles which will attach

particles.

When the motion of the particles is blocked by a hard boundary, the resuhing

accumulation of particles at the boundary is called sediment. Sedimentation describes

the motion of molecules in solutions or particles in suspensions in response to an

external force such as gravity, centrifugal force or electric force. Sedimentation may

pertain to objects of various sizes, ranging from suspensions of dust and pollen

particles to cellular suspensions to solutions of single molecules. Even small

molecules such as aspirin can be sediment, although it can be difficult to apply a

sufficiently strong force to produce significant sedimentation.

Incineration greatly reduces the volume of the sludge and eliminates biohazard

concerns. Such systems require multi-step cleaning of the exhaust gas. However, the

ash is difficult to use due to its high heavy metal content.

Filtration is another physical process and uses a medium such as sand as a final

treatment stage can result in very clear water. Similarly ultrafiltration, nanofiltration

and reverse osmosis are processes which force water through membranes and can

remove colloidal material (very fine, electrically charged particles, which will not

settle) and even some dissolved matter.

Adsorption is one of the widely used physical treatment process. Adsorption on

activated charcoal can removes dissolved chemicals also. Air or steam stripping can

be used to remove pollutants that are gases or low-boiling liquids from water and the

vapours which are removed in this way are also often passed through bed of activated

charcoal to prevent air pollution.

Equaliialion can be used to even out wide variations in flow rates. Some industries

produce different types of effluents (containing different characteristics) at different

interval of lime. Hence, uniform treatment is not possible. In order to obviate this

problem equalization is used.

These are ph} sieal processes to remove the contaminant from the wastewa

8

1.2.2 Chemical Process:

This treatment may remove dissolved substances from wastewater. A chemical

treatment may include any of the following or all:

Disinfection is usually the final process before discharge. In order to destroy the

harmful microorganisms, the effluent treated with some sort of disinfectant. Chlorine,

ozone and ultraviolet light used as a disinfectants.

In chlorination highly toxic cyanides converts in mining and metal finishing

industries into harmless CO2 and N2 by oxidizing them with CI2. In ozonation organic

chemicals destroy by oxidizing them using O3 or H2O2, either alone or in combination

with catalyst or ultraviolet light.

A chemical process commonly used in many industrial wastewater treatment

operations is neutralization. Neutralization consists of the addition of acid or base to

adjust pH levels back to neutrality. Since lime is a base it is sometimes used in the

neutralization of acid wastes. Coagulation consists of the addition of a chemical that,

through a chemical reaction, forms an insoluble end product that serves to remove

substances from the wastewater.

Precipitation is a process in which dissolved metal convert into a solid and settleable

form by precipitation with an alkaline material like sodium or calcium hydroxide.

Dissolved iron or aluminum salts or organic coagulants aid like polyelectrolyte can be

added to help flocculate and settle (or float) the precipitated metal.

Processes such as ion exchange, which involves exchanging certain ions for others,

are not used to any great extent in wastewater treatment. Adsorption may actually be

physical and chemical in nature. The use of activated carbon to adsorb or remove

organics, involves both chemical and physical processes.

Physical and chemical process has drawbacks such as their high cost and formation of

hazardous by products. In most cases chemical treatment increased the dissolved

solids of the water and wastewater.

1.2,3 Biological Process:

In biological treatment processes microorganisms (mostly bacteria) are used, for the

decomposhion of toxic compounds present in wastewaters to form benign stable end

product. Waste is converted to carbon dioxide, water and other end

products. Microbial attack on organic compounds depends on two factors:

I. Steric hindrance: As the substitution on the substrate increases the approach of

microorganisms towards the substrate become difficult.

11. Nature of the substituent on the substrate: Attack of microorganisms on

organic compound is electrophillic in nature, Thus all those groups (chloro,

nitro etc. which decrease the electron density of aromatic ring) makes the

microbial attack difficult and those groups which increases the electron density

on aromafic ring favours the microbial attack on the aromatic ring.

Industries deals with biodegradable materials such as food processing, dairies,

breweries, paper, plastics and petrochemicals etc. use biological treatment. This

method is very useful and removes all types of dissolved substances which are

hazardous to the environment. Biological treatment is divided into two categories viz.

anaerobic and aerobic based on availability of dissolved oxygen.

1.2.3.1 Anaerobic Process:

Anaerobic process means treatment in a closed container in absence of air. UASB

(upflow anaerobic sludge blanket reactor) is most popular among anaerobic process

reactors. For anaerobic process much more efficient reactors have been developed for

UASB reactors (Lettinga et al.. 1993). In some anaerobic (Letlinga et al., 1980) and

anoxic (Green et al., 1994) process like UASB, granular sludge may be developed.

Bui anaerobic process like UASB suffered from odour problem and also the final

products formed, are not completely benign, due to incomplete oxidation in case of

10

anaerobic process. Also these processes are useful only for high organic load but they

can not be designed for lower organic load.

1.2.3.2 Aerobic Process:

The aerobic process of wastewater, the treatment plant often accomplished by means

of the application of conventional activated sludge systems. In recent years, new

technologies are being developed to improve this system. In aerobic process activated

sludge and aerobic granulation technologies are used.

1.2.3.2.1 Activated Sludge Process:

Most common aerobic process is activated sludge process. Activated sludge is a

process dealing with the treatment of sewage and industrial wastewaters (Beychok et

al., 1967). The combination of raw sewage or industrial wastewater and biological

mass is commonly known as mixed Liquor. In all activated sludge plants, excess

mixed liquor is discharged into settling tanks and the treated supernatant is run off to

undergo further treatment before discharge. Part of the settled material, the sludge, is

returned to the head of the aeration system to re-seed the new sewage or industrial

wastewater entering the tank. This fraction of the floe is called return activated sludge

(R.A.S.). It invohes a series of steps- primary treatment, secondary treatment and in

some cases a tertiary treatment may also be used. The effluent from one tank becomes

(he inlluent for the other.

Primary treatment removes suspended impurities. Here, the wastewater is first passed

through a grit chamber, where suspended particles greater than the size of the grit are

removed and the water is allowed to pass into the sedimentation tank. In

sedimentation tank, the remaining suspended particles get settle down due to gravity

and the clear effluent is passed into the aeration tank where air is supplied at the

bottom b}' means of mechanical aerators. This will lead to the efficient mixing of

effluent.

11

Table 1.2: Wastewater Treatment Processes

PHYSICAL CHEMICAL BIOLOGICAL

Flocculation

Sedimentation

Incineration

Filtrarion

Adsorption

Air or Steam Stripping

Equalization

Disinfection

Clilorination

Ozonation

Neutralization

Coagulation

Precipitation

Ion Exchange

Adsorption

Anaerobic Treatment

(UASB)

Aerobic Treatment

I. Activated Sludge

II. Aerobic granulation

12

The effluent from aeration tank becomes influent for secondary clarifier. In secondary

tank, the wastewater is mixed with biological mass (microorganisms) which degrade

the complex dissolved organic compounds into simple compounds CO2 and RjO. In a

sewage or industrial wastewater treatment plant, the activated sludge process can be

used for one or several of the following purpose:

• Oxidizing carbonaceous matter: biological matter.

• Oxidizing nitrogenous matter: mainly ammonium and nitrogen in biological

materials.

• Removing phosphate.

• Driving off entrained gases carbon dioxide, ammonia, nitrogen etc.

• Generating a biological floe that is easy to settle.

• Generating a liquor low in dissolved or suspended material.

But activated sludge process is also associated with a number of disadvantages:

• Large space is required.

• More power is required.

• It has low efficiency.

• Activated sludge process fails with high organic load as well as fluctuations on

organic load.

• It can not be amenable to computer control.

1.2.3.2.1.1 Types of Plants:

There are various types of activated sludge plants (Beychok et al., 1967). These

include:

13

I. Package plants are commonly variants of extended aeration, to promote the 'fit

and forget' approach required for small communities. There are various

standards to assist with their design (Code of Practice, Flows and Loads-2;

British Water, Review of UK and international standards; British Standard,

1983).

II. Oxidation ditches are installed commonly as 'fit and forget' technology, with

typical design parameters of a hydraulic retendon time of 24-48 hours and a

sludge age of 12-20 days.

HI. The costs of deep shaft construction are high. Deep Shaft was developed by

ICI, as a spin-off from their pruteen process. It is Aker Kvaerner Engineering

Services (Deep Shaft Process Technology).

IV. Surface aerated basins may range in depth from 1.5 to 5.0 meters and utilize

motor-driven aerators floating on the surface of the wastewater (Beychok et

al., 1971).

In order to overcome the disadvantages of activated sludge process and to achieve

high efficiency of treatment plants, researchers are continuously searching to find new

processes and found technique which is cell immobilization technology.

1.2.3.2.2 Aerobic Granular Sludge Process or Aerobic Granulation Technology:

Cell immobilization technology can withstand high organic load as well as

fluctuations in organic load. In immobilization technology microbial cells get

immobilized either on a surface or with each other. When immobilized on a surface, it

is called as hiofilm formation and when immobilized with respect to each other, it is

called as aerobic granulation.

In this technique, the light and dispersed sludge is washed out and the relatively

heavy granules are retained by the reactor. This technology has been extensively

reported in sequencing or sequential batch reactor (SBR) and is customized for

treating a wide variety of wastewaters (Beun et al., 1999; Peng et al., 1999; Tay et al.,

2001; Moy et al, 2002: Lin et al.. 2003; Yang et al., 2003; Arrojo et al., 2004; de

14

Kreuk and van Loosdrecht 2004; McSwain et al., 2004; Schwarzenbeek et al., 2004,

2005 and Zhu et al., 2004).

• Sequencing Batch Reactor:

Interest in SBRs was revived in the late 1950s and early 1960s, with the development

of new equipment and technology. Improvements in aeration devices and control have

allowed SBRs to successfully compete with conventional activated systems and it can

be amenable to computer control also.

The unit process of SBR and conventional activated sludge systems are the same. A

report U.S. EPA, (1983) summarized this by stating "the SBR is no more than an

activated sludge system which operates in time rather than in space". The SBR

performs equalization, biological treatment and secondary clarification in single tank

using a timed control sequence.

The SBR is a fill and draw activated sludge system for wastewater treatment. In this

system, wastewater is added to a single "batch" reactor, treated to remove

undesirable components and then discharged. Equalization, aeration and clarification

can all be achieved using a single batch reactor. SBR systems have been successfully

used to treat both municipal and industrial wastewater.

For the investigation of the aerobic granules cultured under alternating aerobic and

anoxic conditions a SBR was operated without the presence of carrier material. It was

observed that physical characteristics of granules play an important role in the

performance of the SBR. The major technical problem encountered in operating

aerobic granular sludge SBR relates to instability of aerobic granules. Filamentous

growth has been commonly observed in aerobic granular sludge in SBR (Tay et al.,

2001; Pan, 2003; McSwain et al., 2004; Wang et al., 2004 and Schwarzenbeek et al..

2005) treating different kinds of effluent wastewaters flow (Moy et al., 2002; Pan,

2003; McSwain et al.. 2004: Tay et al.. 2004: Wang et al.. 2004; Jiang 2005: Hu et al.,

2005 and Schwarzenbeek et al.. 2005).

15

The operation of SBR is based on the till and draw principle, which consists of

following tlve basic steps:

I. Fill step

II. React step

III. Settle step

IV. Draw step

V. Idle step

Design principle of steps of sequencing batch reactor (SBR) is shown in Fig 1.1.

More than one operation strategy is possible during most of these steps. For industrial

wastewater applications, treatability studies are typically required to determine the

optimum operating operation. For most municipal wastewater treatment plants,

treatability studies are not required to determine the operating sequence because

municipal wastewater flow rates and characteristic variations are usually predictable

and most municipal designs will follow conservatives design approaches.

During Fill step influent wastewater is added into the reactor to the biomass which is

already present in the SBR. This step is characterized by no mixing or aeration,

meaning that there will be a high substrate (food) concentration when mixing begins.

The conditions of fill step favours organisms that produce internal storage products

during high substrate conditions. This step may be compared by using "selector"

compartments in a conventional activated sludge system to control the F: M ratio.

In react step mixing and aeration occurs. Mixing is classified by adding influent

organics with the biomass. which initiates biological reactions. During mixing,

microbes biologically degrade the organics and use residual oxygen or alternative

electron acceptors. Anaerobic conditions can also be achieved during the mixing

phase. Aeration is classified by aerating the contents of the reactor to begin the

aerobic reactions completed in the react step. Aeration can reduce the aeration time

required in the react step. The biological reactions initialized during aeration are

completed.

16

The settling stage depends upon the setthng property of sludge. During this stage the

sludge formed by the bacteria is allowed to settle to the bottom of the reactor. In the

initial stage of settling results in a clearer effluent and a more concentrated settled

sludge. In SBR there are no influent or effluent currents to interfere with the settling

process as in a conventional activated sludge system. The sludge is allowed to settle

until clear water is on the top 20%-30% of the reactor contents.

The draw steps use a decanter to remove the treated effluent, which is the primary

distinguishing factor between different SBR manufactures. There are floating

decanters, and fixed decanters. Floating decanters offer several advantages over fixed

decanters.

The idle step occurs between the draw and the fill steps, during which treated effluent

is removed and influent wastewater is added. The idle step is often not a part of

aerobic granulation. The length of the idle step varies depending on the influent flow

rate and the operating strategy. Equalization is achieved during this step if variable idle

times are used. Mixing is the condition of biomass and sludge wasting can also be

performed during the idle step, depending on the operating strategy.

Information on the formation of granules under aerobic conditions is still a subject to

discussion (Morgenroth et al, 1997; Beun et al, 1999 and Etterer and Wilderer, 2001).

Several investigations have focused on the floe size distribution and the development

of aerobic granulations (Li and Ganczarczyk 1990; Dangcong et al., 1999 and Tay et

al., 2001). Aerobic granulation is a gradual process which involves progression from

suspended sludge to aggregates and further to aerobic granules with a regular shape

and compact structure (Moy et al., 2002; Tay et al., 2001, 2002 and Liu et al., 2005) or

aerobic granulation is a gradual process from seed sludge to compact aggregates, to

granular sludge and finally to mature granules (Tay et al, 2001a). Aerobic granulation

is believed to be a microbial self-immobilized process that is driven by selection

pressure in SBR (Kim et al, 2004; Qin et al., 2004; Hu et al. 2005 and Liu et al.,

2005a).

17

Fill

US.

Idle

Draiv

- - W

React

Settle

Decant

Aeration/ niixias

Fig 1.1: Design principle of steps of sequencing batch reactor (SBR).

18

•* Aerobic Granule:

Granules making up aerobic granular activated sludge are to be understood as

aggregates of microbial origin, which do not coagulate under reduced hydrodynamic

shear and which settle significantly faster than activated sludge floe (de Kreuk et al.,

2005). Aerobic granules have gained a strong research interest in recent years (Jiang

et al., 2002; Moy et al., 2002; Tay et al., 2002a-d and Jiang et al., 2003).

I. Characteristics of Aerobic Granules:

• Regular, smooth, round shape and a clear outer surface.

• Have compact and stronger microbial structure.

• Be visible as separates entities in the mixed liquor during both the mixing and

the settling phase.

• Have a high active biomass retention and excellent settelability.

• Be capable to withstanding high flow rates.

• Be able to withstand high organic loading rates and fluctuations in organic

loads.

• Be less vulnerable than the suspended sludge to the toxicity of organic

chemicals and heavy metals in \\astewater. The excellent settleability of

aerobic granules simpliiles the separation of treated effluent from the granular

sludge.

Aerobic granules have been successfully cultivated with a wide variety of organic

substrates in SBR, including glucose, acetate, ethanol, phenol, particulate organic

matter-rich wastewater and both simulated and real municipal wastewater

(Morgenroth et al.. 1997; Beun et al.. 1999; Peng et al., 1999; Tay et al.. 2002a; Jiang

et al.. 2002: Liu et al., 2003; Schwarzenbeck et al.. 2003; Pan 2003 and Arrojo et al.,

2004). Nitrifying and phosphorus accumulating granules have also been developed

(Tay et al.. 2002b: I.in et al.. 2003 and Tsuncda et al.. 2004).

19

The biological system for wastewater treatment depends significantly on the active

biomass concentration, the overall biodegradation rates, the reactor configuration and

the feeding rates of the substrates and oxygen. By using aerobic granule or sludge in

ways that allow high conversion rates and efficient biomass separation to minimize

the reactor volume, process efficiency of large scale treatments plants can be

improved. Treating capacities can be easily varied to accommodate varying loading

rates, wastev^ater composition and treating goals by bioaugmentation with specifically

developed granules.

1.2.3.2.2.1 Factors Affecting Aerobic Granulation:

The factors involved in aerobic granulation in SBR, includes substrate composition,

organic loading rate, hydrodynamic shear force, feast famine regime (availability and

unavailability of food), feeding strategy, dissolved oxygen, reactor configuration,

solid retention time, cycle time, settling time and exchange ratio (Liu et al., 2005a).

The major selection pressures responsible for aerobic granulation are identified as the

settling time and exchange ratio.

• Substrate Composition:

Aerobic granules have been successfully formed with several substrates including

glucose, acetate, ethanol, phenol and synthetic wastewater. The microstructure and

species diversity of granules are related to the type of carbon source. The glucose fed

aerobic granules have exhibited a filamentous structure and acetate fed granules has a

non iilamenlous structure (Tay et al., 2002 and Wang et al.. 2004). Filamentous

granules have also been found in phenol fed aerobic granules (Jiang et al., 2005).

• Organic Loading Rate:

Granules have the ability to retain active biomass. Granule can handle high organic

loading rates (OLR). Most wastewater treatment systems have relatively low OLRs.

Howcx'cr. little is known about the application of high OLRs to aerobic granules

20

grown in sequencing batch reactors, studies employed OLRs below 7.5 kg COD m'

d'' (Beun et al., 1999 and Tay et al., 2001a). Aerobic granules can be developed over

a very wide range of organic loading rates as 2.5-15.0 kg COD m~ d~' (Moy et al.,

2002 and Liu et al., 2003), while nitrifying granules can also be formed over a very

wide range of ammonia-nitrogen loadings (Yang et al., 2003; Qin et al., 2004c and

Tsuneda et al., 2004). Aerobic granulation in SBR is independent of substrate

concentration (Liu et al., 2003), but the kinetics behaviour of aerobic granules is

related to the applied organic loading (Moy et al., 2002 and Liu et al., 2003).

Although the effect of OLRs rate on the formation of aerobic granules is insignificant,

the physical characteristics of aerobic granules depend on the OLRs. The mean size of

aerobic granules is increased with the increase in OLRs. The physical strength of

aerobic granules is decreased with the increases of OLRs.

• Hydrodynamic Shear Force:

Evidence shows that a high shear force favours the formation of aerobic granule. It is

determined that aerobic granules could be formed only at a threshold shear force

value in terms of superficial upflow air velocity above 1.2 cm s"' in a column SBR.

Due to high hydrodynamic force regular, spherical and compact aerobic granules

were developed. Applied shear force is proportionally related to density and strength

of granules. These findings suggest that the hydrodynamic shear force is determining

factor for the structure of aerobic granule. At high shear force the stimulated

production of extracellular polysaccharide (EPS) was also observed (Tay et al..

2001b: Liu and Tay. 2002). It is also known that EPS can mediate both cohesion and

adhesion of cells and play a crucial role in maintaining the structural integrity in a

community of immobilized cells (Liu et al.. 2004b). It is reported that shear force was

closely associated with the production of EPS. The production of EPS was found to

be related to the stability of aerobic granules. The EPS content normalized the protein

content, increased with the shear force estimate bacteria to secrete more EPS. Shear

force induced production of EPS has also been observed in biofilms. The enhanced

21

production of EPS at high shear force can contribute to the compact and stronger

structure of aerobic granules. Although, the hydrodynamic shear force is not a

primary inducer of aerobic granulation in SBR (Liu and Tay, 2002).

• Settling Time:

The effect of settling time on aerobic granulation in SBR is studied by Qin et al.,

(2004a, b). In SBR, wastewater is treated in successive cycles each lasting for few

hours. At the end of the every cycle, the aeration is stopped and the biomass is

allowed to settle before the effluent is withdrawn. The settling time is a major

hydraulic selection pressure on microbial community. A short settling time

preferentially selects for the growth of fast settling bacteria and the sludge with poor

settleability is being washed out. It is found that aerobic granules v/ere successfully

cultivated and became dominant. The production of EPS was stimulated and the cell

surface hydrophobicity improved significantly at short settling times. A short settling

time has been commonly employed to enhance aerobic granulation in SBR (Jiang et

al.. 2002; Lin et al., 2003; Liu et al., 2003; Yang et al., 2003; Wang et al. 2004 and

Hu et al., 2005). The aerobic granulation is driven by selection pressure and the

formation and characteristic of the granules may be controlled by manipulating the

selection pressure. The selection of optimal settling time is very important in aerobic

granulation. Granule with excellent settling properties is essential for the effective

functioning of biological systems treating wastewater.

• Feast-Famine Regime:

In SBR, the aeration period actually has two phases: a degradation phase in which the

substrate is degraded to a minimum, followed by an aerobic starvation phase in which

the external substrate is no longer available. Short feeding periods must be selected to

create alternate feast and famine periods (Beun et al, 1999). Microorganisms in SBR

are subjected to a periodic feast and famine regime, called periodic starvation (Tay et

al.. 2001a). Under the periodic feast-famine conditions, bacteria become more

22

hydrophobic and high cell hydrophobicity in turn facilitates microbial aggregation

(Bossier and Verstraete 1996; Tay et al., 2001a and Liu et al., 2004a). When bacteria

are subjected to a periodic feast-famine regime, microbial aggregation could be an

effective strategy for cells against starvation. In fact, the periodic feast-famine regime

in SBR can be regarded as a kind of microbial selection pressure that may alter the

surface properties of cells. However, more recent research showed that aerobic

granules could not be successfully developed if the settling time in SBR was not

properly controlled, even though a periodic feast-famine regime was present (Qin et

al., 2004a, b), while negative effects of nutrient starvation on the surface properties of

aerobic granules in terms of cell hydrophobicity and the content of EPS were also

observed (Zhou, 2004). This may imply that the periodic feast-famine regime could

favour aerobic granulation, but so far there is no proof to show that starvation acts as

an inducing force of aerobic granulation in SBR.

• Feeding Strategy:

During the SBR operation a periodic starvation occurs. This starvation period has

been shown to have a profound effect on cell hydrophobicity, which is the key factor

that affects aerobic granulation. The cell surface hydrophobicity was proportionally

related to starvation time in SBR. Different filling time were applied to SBR reactors

that is intermittent feeding was developed by McSwain et al, (2003) an operating

strategy to enhanced aerobic granulation. A feast famine cycle or pulse feeding of

SBR favoured the formation of compact and dense aerobic granules. Bacteria became

more hydrophobic and this in turning facilitated microbial adhesion and aggregation

under starvation condition.

• Dissolved Oxygen:

Dissolved oxygen (DO) concentration is an important variable that influences the

operation of aerobic wastewater treatment systems. Aerobic granules formed at a DO

concentration as low as 0.7-1.0 mg f' in a SBR (Peng et al., 1999). In addition

23

aerobic granules have been successfully developed at DO concentrations of 2-6 mg

r ' (Yang et al., 2003; Qin et al., 2004a and Tsuneda et al.. 2004). It appears that DO

concentration is not a decisive parameter in the formation of aerobic granulation.

• Solids Retention Time:

Sludge age or solids retention time is a variable of activated sludge process. In SBR

the SRT up to 30 days had no significant influence on aerobic granulation (Pan,

2003). Aerobic granulation has never been reported in activated sludge processes

operated in an extremely wide range of SRT, from several hours to hundreds of days

(Liu and Tay, 2002). So SRT would not be an inducing force for aerobic granulation.

• Reactor Configuration:

In almost all; reported cases, aerobic granules produced in column type upflow

reactors. Reactor configuration has an impact on the flow pattern of liquid and

microbial aggregates in the reactors. The column type upflow reactor and completely

mixed tank rectors (CMTR) have very different hydrodynamic behaviour in terms of

interaction between flow and microbial aggregates. In column reactors the air and

liquid upflow can create a relatively homogenous circular flow and localized

vortexing along the axis of reactor and microbial aggregates are constantly subjected

to hydraulic attrition. The circular flow apparently forces the microbial aggregates to

adapt a regular granular shape that has a minimum surface free energy (Liu and Tay.

2002)

In a column type upflow reactor a high ratio of reactor height to diameter (H/D) can

ensure a longer circular flow trajectory which in turns provides a more effective

hydraulic attrition to microbial aggregates. However, a high H/D ratio may improve

oxygen transfer and could also result in a reactor with a small footprint (Beun et al..

2002). Recent research revealed that aerobic granules could be developed in SBRs

with diflerenl H/D ratios (Pan, 2003). In CMTRs microbial aggregates stochasticall}'

move with dispersed flow in all directions. Thus, microbial aggregates are subjected

24

to varying localized hydrodynamic shear force, upflow trajectories and random

collisions. Under such conditions, only floes with irregular shape and size instead of

regular granules occasionally form. For practical applications, the SBR should have a

high H/D ratio to improve selection of granules by the difference in settling velocity.

A high H/D ratio and the absence of an external settler results small footprints in a

reactor.

• Exchange Ratio:

The exchange ratio in SBR is defined as the liquid volume withdrawn at the end of

the given settling time over the total reactor working volume. For column SBR with

the same diameter, the exchange ratio is proportionally correlated to H, the height

from the discharging port to the water surface. Keeping the settling time constant for

5 min Wang et al., (2004) studied aerobic granulation in SBR run at different

exchange ratio in the range of 20-80%. A larger exchange ratio is associated with a

higher H. The fraction of aerobic granules in the total biomass was found to be

proportionally related to the exchange ratio. At a fixed exchange ratio of 75%, Zhu

and Wilderer, (2004) successfully developed aerobic granules. These results provide

experimental evidences that aerobic granulation is highly dependent on the exchange

ratio of SBR.

• Cycle Time:

The duration of the substrate oxidation reaction time represents the SBR cycle time.

C)cle time is interrelated to the hydraulic retention time (HRT). Microbial growth can

be hindered by an insufficient reaction time for microorganisms to breakdown

substrates, if the SBR is run at an extremely short cycle time. As a result, the sludge

loss due to hj'draulic washout from the system cannot be compensated by the growth

of bacteria. Hydrolysis of the biomass would eventually cause a negative effect on

microbial aggregation, if the cycle time is kept much longer than that required for a

biological reaction (Tay et al.. 2002b; Chen et al.. 2003: Pan et ah. 2004 and Zhou.

2004). The cycle time of SBR should be short to suppress biomass hydrolysis, but

25

long enough for biomass growth and accumulation in the system. Recent research

clearly demonstrated that, for SBRs operated at the optimum cycle time determined

previously. Qin et al., (2004a, b) found during his research work that at longer settling

time mixture of aerobic granules and suspended solids developed but aerobic granules

are cultivated at a settling time of less than 5 minutes. This implies that cycle time is

not a decisive factor for aerobic granulation in SBR.

From the above discussion it is found that aerobic granulation in SBR could fail

without the proper control of settling time or exchange ratio. Settling time and

exchange ratio act as the main hydraulic selection pressure which can induce aerobic

granulation in SBR. Liu et al., (2005a) suggest that aerobic granulation in SBR is

mainly initiated and driven by a short settling time or a high exchange ratio that

selects bioparticles according to their settling velocity.

1.2.3.2.2.2 Mechanism of Aerobic Granulation:

A number of conditions are needed to be met to form aerobic granules from

microorganisms. The physical, chemical and biological forces contributing to

granulation need to be viewed in combination. Aerobic granulation consists of the

following steps:

• Physical movement to initiate bacteria to bacteria contact. The factors

involved in this step are hydrodynamic shear force, diffusion mass transfer,

gravity, thermodynamic effects and cell mobility.

• Stabilization of the multi cell contact resulting from the initial attractive

forces. These attractive forces are physical (e.g. Van der Waals forces,

opposite charge attraction, hydrophobicity, filamentous bacteria that can

bridge individual cells), chemical and biological forces including cell surface

dehydration, cell membrane fusion, signaling, and collective action in bacterial

community.

• Maturation of cell aggregation through production of extracellular polymer,

growth of cellular cluster, metabolic change and environment induced genetic

26

effect that facilitate the cell to cell interaction and result in a highly organized

microbial structure.

• Shaping of the steady state three dimensional structure of microbial aggregate

by hydrodynamic shear forces.

Although mechanism and models for aerobic granule have been described, they do

not provide a complete picture of granulation process.

1.2.3.2.2.3 Advantages of Aerobic Granulation Technology:

Aerobic granules in SBR present have several advantages compared to conventional

activated sludge process such as:

• Stability and Flexibility:

The SBR system can be adapted to fluctuating conditions with the ability to

withstand shock and toxic loadings.

• Excellent Settling Properties:

A smaller secondary settler will be necessary, which means a lower surface

requirement for the construction of the plant.

• Good Biomass Retention:

Higher biomass concentrations inside the reactor can be achieved and higher

substrate loading rates can be treated.

Inside the granules aerobic and anoxic zones are present to perform simultaneously

different biological processes in the same system (Beun et al., 1999). The cost of

running a wastewater treatment plant working with aerobic granular sludge can be

reduced by at least 20% and space requirements can be reduced by as much as 75%

(de Kreuk et al., 2004). The feasibility study showed that the aerobic granular sludge

technology seems very promising (de Bruin et al., 2004).

27

1.2.3.2,2.4 Application of Aerobic Granular Technology:

Recent studies have shown that aerobic granulation technology could be applied for

high strength organic wastewater treatment (Moy et al., 2002) and simultaneous

organics, nitrogen removal (Yang at al., 2003) and toxic wastewater treatment (Jiang

et al., 2002). This indicates that aerobic granulation technology have capacity to treat

municipal and industrial wastewater both.

• High Strength Organic Wastewater Treatment:

Granulation of the sludge can lead to high biomass retention in the reactor because of

the compact and dense structure of the granules. Aerobic granules have ability to

sustain high organic loading rates increases in organic loading only after the COD

removal efficiencies has stabilized (Moy et al., 2002). Aerobic granules were able to

sustain the maximum organic loading rate of 15 kg COD m" d"' while removing more

than 92% of the COD.

At low loading filamentous bacteria dominated the granules initially exhibited a fluffy

loose morphology and evolved in to smooth irregular shapes as folds, crevices and

depressions at higher loading. For better diffusion and penetration of nutrient into the

interior of granules these irregularities were thought to allow. The higher substrate

concentration existed in the bulk wastewater at hiaher loading mass transfer of

nutrients was also enhanced. These factors enabled the aerobic granules to sustain

high organic loadings rates without compromising the granules integrity.

• Phenolic Wastewater Treatment:

Various industries such as oil refineries, petrochemical plants, coke conversion,

pharmaceuticals and resin industries produces many toxic substances as their effluent,

phenol being one of them. Phenol concentration of up to 10,000 mg 1' has been

reported In many industrial wastewaters (Fedorak and Hrudey. 1988). The phenol

degrading aerobic granules shows an excellent ability to degrade phenol. For an

28

influent phenol concentration of 500 mg 1"' a stable effluent phenol concentration of

less than 0.2 mg f' was achieved in the aerobic granular sludge reactor. Granular

sludge is less susceptible to toxicity of phenol because much of the biomass in the

granules is not exposed to the same high concentration as present in the wastewater.

The high tolerance of aerobic granules to phenol can be exploited in developing

compact high rate treatment systems for wastewater loaded with a high concentration

of phenol. Aerobic granules appeared to be highly tolerant to toxic heavy metals also.

• Nitrogen Removal:

Complete nitrogen removal involves nitrification and denitrification. Nitrites and

nitrate produced from nitrification are reduced to gaseous nitrogen by denitrifiers.

Researchers have investigated the simultaneous removal of organics and nitrogen by

aerobic granules. Increased N/COD ratio led to significant shifts among the three

populations within the granules. Enhanced activities of nitrifying and denitrifying

population were achieved in granules developed at high substrate N/COD ratio. It

appears that complete organics and nitrogen removal can be efficiently and stably

achieved in a single granule based SBR.

29

1,3 Water Quality Parameters:

A number of parameters are considered to check the quality of water. These

parameters as per APHA, (J 998) are:

pH

Conductivity

Chemical Oxygen Demand (COD)

Biochemical Oxygen Demand (BOD)

Dissolved Oxygen (DO)

Total Solids (TS)

Total Dissolved Solids (TDS)

Total Suspended Solids (TSS)

Volatile Suspended Solids (VSS)

Settled Sludge Volume

Sludge Volume Index (SVI)

Concentration of Organic Compounds

The UPSH and ISI have set standards for various water quality parameters. These are

listed in the Table 1.3. Also WHO, (1993) has given the standard concentrations of

various components that may be present in the drinking water (Table 1.4).

30

Table 1.3: Standards of Water Quality Parameters

PARAMETERS

Colour, odour, taste

Inorganic Chemicals

pH

Specific conductance

Dissolved oxygen (D.O.)

Total dissolved solids

Suspended solids

Chloride

Sulphate

Cynide

Nitrate+nitrite

Fluoride

Phosphate

Sulphide

Ammonia

Boron

Calcium

UPSH STANDARD

Colourless, odourless,

tasteless

6.0-8.5

300 mmho cm"

4.0-6.0 (ppm)

500

5.0

250

250

0.05

<10

1.5

0.1

0.1mgL"'(PPb)

0.5

1.0

100

ISI STANDARD (IS:

2296-1963)

"

6.0-9.0

-

3.0

-

-

600

1000

0.01

-

3.0

-

-

-

-

_

31

• Magnesium

Arsenic

Barium

Cadmium

Chromium (VI)

Copper

Iron (filterable)

Lead

Manganese (filterable)

Mercury

Selenium

Silver

Uranium

Zinc

Organics

COD

30

0.05

1.0

0.01

0.05

1.0

<0.3

<0.05

<0.05

0.001

0.01

0.05

5.0

5.5

4.0

0.2

0.05

0.01

0.05

Carbon CHCI3 extract (CCE) 0.15

Methylene blue active

substances

Phenols ;

Pesticides (total)

0.5

0.001

0.005

0.005

32

Polycyclic aromatic 0.2 ppb (0.002 ppm)

hydrocarbons (PAH)

Surfactants

Radioactivity^

200

Gross beta

Radium-226

Strontium-90

1000 pcL '

3 pc L'

lo pc L" <5000

Bacteriological Parameters

Coliform cells/100 ml 100

Total bacteria count/ 100 ml 1*10

The official journal of European Committees, L 229, pp. 16-21 (1980). Quotes figures close to

those of USPH. The ISI values are considerably on the high side, which remain open to question

and these do not cover all the parameters.

All the units, except otherwise mentioned and pH, specific conductance and radioactivity, are in

ppm, i.e. parts per million or mg f'. The radioactivity units are in picacuries I'', i.e. 10"'"curies f'

(2.2 disintegrations min"' I"').

33

Table 1.4: Standard Concentrations of Drinking Water Components

COMPONENTS CONCENTRATION ( G L ')

"AS \0

B 300

Cd 3

Cr 50

Cu 2000

F 1500

Pb 10

Mn 500

Hg 1

NO3" 50

NO2" 3

Se 10

Organics

Benzene 10

Benzo(a)pyrene 0.7

i r E 70

NTA 200

34

Pesticides

Atrazine 2

Lindane 2

2.4-D 30

DDT 2

Disinfection Byproducts

Monochloramine 3000

Di- and trichloroamine 5000

Chloroform 200

35

TERMINOLOGY USED IN THIS CHAPTER:

Hydraulic Retention Time:

The Hydraulic retention time (HRT) is a measure of the average length of time in

which soluble compound remains in a constructed reactor.

Volume of aeration tank HRT= —•

Influent flow rate

In which 'volume is in m ' SI unit and 'Influent flow rate is in m7h' SI unit. So SI Unit

of HRT is same as that of time. It is usually expressed in hours (or sometimes days).

Hydraulic retention time is an important parameter in many wastewater treatment

processes. To optimize reactor performance, a proper HRT should be judiciously

selected and carefully maintained (Fang and Yu 2000, 2001). The HRT must be

shorter than the inverse of the maximum growth rate of suspended bacteria to achieve

high biomass concentration in the reactors and to ensure that the carrier surfaces are

completely colonized by heterotrophic biofilms (Tijhuis et al., 1994). In case of

activated sludge systems values of HRT range vary for aeration basins domestic

wastewater (Ardern and Lockett, 1914). However, in UASB, a short HRT and high

upllow velocity were employed to separate the lighter and heavier sludge fractions

(0'Flaherty et al., 1997). The SBR cycle time represents the frequency of solids

discharged through effluent withdrawal which is related to the HRT at a given

exchange ratio. The SBR cycle time is a main factor which can serve as a hydraulic

selection pressure on the microbial community in the system. The effect of hydraulic

selection pressure on the development of nitrifying granules in SBR has also been

investigated. Microbial acti\'ity, production of cell polysaccharides and improvement

of cell hydrophobicity stimulates by short time cycle. This hydraulic selection

pressure induced microbial changes which favours the formation of nitrifying

bacteria.

36

Effluent:

Effluent is an outflow of water from a natural body of water or from a man-made

structure. Effluent in the man-made sense is generally considered to be water

pollution, such as the outflow from a sewage treatment facility or the wastewater

discharge from industrial facilities. In the context of wastewater treatment plants,

effluent that has been treated is sometimes called secondary effluent or treated

effluent. This cleaner effluent is then used to feed the bacteria in biofilters.

Liquid waste flowing out of a factory, farm, commercial establishment or a household

into a water body such as a river, lake, lagoon and sewer system is effluent.

Sludge:

Sludge is the residual semi-solid material left by industrial water treatment or

wastewater treatment processes. Excess solids from biological processes such as

activated sludge can be referred to as sludge, although more often called biosolids.

Industrial wastewater solids are also referred to as sludge, whether generated from

biological or physico-chemical processes. Surface water plants also generate sludge

made up of solids removed from the raw water.

Disinfectant:

Disinfection is usually the final process before discharge. The effluent from SBR is

treated with some sort of disinfectants, in order to destroy the harmful (pathogenic)

microorganisms, i.e. disease-causing germs. The object is to reduce the number of

harmful ones to appropriate levels for the intended use of the receiving water.

The most commonly used disinfectant is chlorine, which can be supplied in the form

of a liquefied gas which has to be dissoh'ed in water or in the form of an alkaline

solution called sodium hypochlorite (NaC104). Chlorine is quite effective against

most bacteria, but a rather high dose is needed to kill viruses, protozoa and other

forms of pathogen. Hypochlorite is safer, but still creates problems and the first two

problems. Chlorine is very toxic to aquatic organisms, this can be dealt with by

37

adding sulphur dioxide (SO2), a liquefied gas or sodium sulphite or bisulphite

(solutions) to neutralize chlorine. The products are nearly harmless chloride and

sulphate ions. It is hazardous to store and handle.

A more powerful disinfectant is ozone (Oj). Ozone is too unstable to store, while

chlorine can be dosed at a high initial concentration so that some of it remains in the

water for a considerable time, ozone is consumed very rapidly and leaves no residual.

It may also produce some chemical by-products, but probably not as harmful as those

produced by chlorine.

The other commonly used disinfectant is ultraviolet light. The water is passed through

banks of cylindrical, quartz-jacketed fluorescent bulbs. Anything which can absorb

the light, such as fouling or scale formation on the bulb's surfaces or suspended

matter in the water, can interfere with the effectiveness of the disinfection. Some

materials, such as iron and some other compounds can absorb some of the light.

Ultraviolet disinfection is becoming more popular because of the increasing

complications associated with the use of chlorine.

Biodegradation:

Biodegradation is the process by which organic substances are broken down by the

enzymes produced by living organisms. The term is often used in relation to ecology,

waste management and environmental remediation (bioremediation). Organic

material can be degraded aerobically with oxygen or anaerobically without oxygen. A

term related to biodegradation is biomineralisation. in which organic matter is

con\erted into minerals. Biosurfactant, an extracellular surfactant secreted by

microorganisms enhances the biodegradation process.

This thesis deals with the development of biodegradation methods for the removal of

three organic xenobiotic compounds namely phenol, para nitro phenol and 2,4-

dinitrophenol.

38

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42

43.Pan S. (2003). Inoculation of microbial granular sludge under aerobic

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44

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45

CHAPTER 2

BIODEGRADATION OF PHENOL BY

AEROBIC QRANULATION TECHNOLOGY

IN SEQUENCING BATCH REACTOR

Biodegraclation of Phenol by Aerobic Granulation Technology in

Sequencing Batch Reactor

In wastewater, bacteria play an important role in the degradation of phenol. Out of the

total population present in water only a small percentage of microbes are capable of

utilizing phenol. However, repeated phenol exposure may result in its accumulation.

Microbes convert phenol into carbon dioxide in aerobic condition and under

anaerobic condition microbes convert phenol into carbon dioxide or methane. In the

biodegradation of phenol, benzoate and acetate are formed as the intermediates.

A number of factors such as phenol concentration, temperature, sunlight, the presence

of other nutrients are required for bacterial growth. The presence of other pollutants

and microbial abundance also affect the contribution of microbes to the overall rate of

degradation.

A brief introduction of phenol including properties, uses and health hazard

information are given below. D '

2.1 PHENOL:

Phenol is used to refer any compound that contains a six member aromatic ring,

bonded directl)' to a hydroxyl group (-011).

Phenol is a constituent of coal tar and is formed during the natural decomposition of

organic material. Phenols are found in the natural world, especially in the plant

kingdom. Phenol can be made from the partial oxidation of benzene, the reduction of

benzoic acid and by the cumene process or by the Raschig-Hooker process. It can also

be formed as a product of coal oxidation. The most common method of producing

phenol is from cumene (isopropyl benzene). Phenol is also produced from

chlorobenzene and toluene. The industries in which phenol is produce as one of the

product are listed below: •

46

Coal gasification, coke conversion, pharmaceuticals, pesticides, fertilizers, dye

manufacturing, resin manufacturing, petroleum refinery, pulp and paper etc.

2.1.1 Structure:

Phenol (CgHjOH) molecular structure and resonance structure are given in Fig 2.1

and Fig 2.2. In phenol, there is an interaction between the delocalized electrons in the

benzene ring and one of the lone pair on the oxygen atom. This has an important

effect on both the properties of the ring and the -OH group. One of the lone pair on

the oxygen overlaps with the delocalized ring electron system. The donation of the

lone pair of oxygen into the ring system increases the electron density around the

ring. That makes the ring much more reactive than it is in benzene itself

2.1.2 Appearance:

Pure phenol consists of white or clear acicular crystals. The crystals are often rather

wet and discoloured. On exposure to air and light, phenol assumes a pinkish or

reddish discolouration, this discoloration is accelerated by the presence of alkalinity

or impurities.

2.1.3 Odour:

Phenol has a characteristic sweet, medicinal or tar-like odour and it is a significant

component which contributes to the aroma of Islay Scotch whisky (Brossard, 2008).

The air odour threshold concentration for phenol is 0.04 parts per million (ppm) of

air.

2.1.4 Physical Properties:

• Molecular Weight: 94.11.

Boiling Point (at 760 mm Hg): 181.7" C (359.1° F).

• Melting Point: 43* C (109.4" F).

47

OH

A, Fig 2.1: Structure of phenol (CeHjOH).

o

Fig 2.2: Resonace structure of phenol.

48

Specific Gravity (water = 1): 1.07 at 20° C (68° F).

Density: 1.07gmcm'\

Vapour Density: 3.24.

Vapour Pressure at 35° C (77° F): 0.35 mm Hg.

Solubility: Soluble in water and benzene. Highly soluble in alcohol,

chloroform, ether, volatile and fixed oils, glycerol, carbon disulfide, petroleum

and aqueous alkali hydroxides. But it is almost insoluble in petroleum ether.

Solubility in Water: 8.3gm/100 ml (20° C).

Acidity (pKa): 9.95. Phenol is slightly acidic. Compared to aliphatic alcohols,

phenol shows much higher acidity because of the resonance stabilization of the

phenoxide anion by the aromatic ring. (John 2"'' ed.). Increased acidity is the

result of orbital overlap between the lone pairs of oxygen and the aromatic

system (Jim, 2007). It has been recently shown that only about 1/3 of the

increased acidity of phenol is due to inductive effects, with resonance

accounfing for the rest (Silva, 2009).

Dipole Moment: 1.70 D.

2.1.5. Chemical Properties:

2.1.5.1 Reactivity:

• Conditions Contributing to Instability: Heat, flames or sparks.

• Incompatibilities: Contact between phenol and strong oxidizers (especially

calcium hypochlorite), acids and halogens should be avoided.

• Hazardous Decomposition Products: Toxic gases (such as carbon monoxide)

may be released in a fire involving phenol.

• Special Precautions: Liquid phenol attacks rubber, coating and some forms of

plastic. Hot liquid phenol attacks aluminum, magnesium, lead and zinc metals.

49

2.1.5.2 Flammability:

The National Fire Protection Association has assigned a flammability rating of 2

(moderate fire hazard) to phenol.

Flash Point: 79° C (175° F).

• Auto ignition Temperature: 715° C (1319° F).

• Flammable Limits in Air (percent by volume): Lower 1.7 and upper S.6.

2.1.5.3 Hazards:

Phenol can pose a severe health hazard and should be handled with extreme care.

When heated, phenol produces flammable vapours that are highly toxic and

explosive.

2.1.6 Uses of Phenol:

• Used as a bonding resin, peptizing agent, blocking agent, intermediates,

preservative, an agent for relieving itching, disinfectant for septic wounds and

as a cauterizing agent.

• Used in synthesis of thermosetting phenolic resins, epoxy, polycarbonate,

phenoxy and polysulfone and in synthesis of caprolactam for use in nylon 6

fibers, plastics and films.

• Used in synthesis of bisphenol-a, adipic acid, alkyl phenols, agricultural

chemicals, intermediates, pharmaceuticals, rubber, plasticizers, antioxidants

and curing agents.

• Used during solvent refining of lubrication, in the synthesis of stabilizers and

preservatives for dyes, perfumes and fungicides.

• Used in medicine for pneumococcal polysaccharide vaccine, in the

manufacture of fertilizer, coke, illuminating gas, lampblack, paints, paint

removers and asbestos goods.

50

• Used in veterinary medicine and gastric anesthetic. Phenol has an internal

antiseptic property. It is also the active ingredient in some oral analgesics such

as Chloraseptic spray.

• Used in the production of drugs (it is the starting material in the industrial

production of aspirin), herbicides and synthetic resins.

• Used in cosmetic surgery as an exfoliant, use in the preparation of cosmetics

including sunscreens (Svobodova et al., 2003), hair dyes and skin lightening

preparations (DeSelms et al., 2008). Compounds containing phenol moieties

can be used to prevent ultraviolet light-induced damage to hair and skin.

2.1.7 Health Hazard Information:

ACGIH, 1991; NIOSHREL, 1992; ACGIHTLV, 1994 and OSHAPEL, 1994 limit the

permissible exposure of phenol: 5ppm for a period of 8-10 hours of work.

2.1.8 Route of Exposure:

Exposure to phenol can occur through inhalation, ingestion, through eye and

absorption through the skin (Sittig, 1991).

2.1.9 Health Effects of Phenol:

Phenol and its vapours are corrosive to the eyes, skin and the respiratory tract

(Budavari, 1996). Repeated or prolonged skin contact with phenol may cause

dermatitis. Inhalation of phenol vapours may cause lung edema (Budavari, 1996)

dysrhythmia, seizures and coma (Warner et al., 1985). It is also a mutagen and there

is some evidence that phenol may be a reproductive hazard.

2.1.9.1 On Animals:

In animals, the predominant effects of acute toxicity are exerted on motor centers in

the spinal cord, which induces marked twitching and severe convulsions. Following

absorption of a toxic dose, the heart rate llrst increases and then decreases or become

51

irregular, the blood pressure initially slightly rises and then falls noticeably. There

may be salivation and marked dyspnea and the body temperature usually decrease

(Clayton and Clayton, 1982). Prolonged oral or subcutaneous administration of

phenol to animals can cause damage to the lungs, liver, kidneys, heart and

genitourinary tract. Prolonged inhalation of vapour concentrations in the range of 30

to 60 ppm causes respiratory difficulties, lung damage, loss of weight and paralysis

(Clayton and Clayton, 1982). Crystalline or concentrated aqueous phenol causes

almost instantaneous white opacification of the corneal epithelium in contact with

rabbit eyes, scarring of the conjunctiva and opacity of the cornea occurred later.

When subconjunctivally 5% phenol injected in rabbit induced glaucoma (Grant,

1986). In the case of rats an increase in leukemia and lymphomas has been reported

when there is 2,500 ppm of phenol in drinking water. Phenol may act as a nonspecific

irritant to promote the development of tumours when it is repeatedly applied in large

amounts to the skin (Hathaway et al., 1991).

2.1.9.2 On Humans:

Skin is a primary route of entry for phenol into the body (Parmeggiani, 1983 and

Hathaway et al., 1991). Systemic absorption causes central nervous system

impairment, liver and kidney damage, local effects include irritation of the eyes, skin

and mucous membranes (Hathavk'ay et al., 1991). An oral dose of 1 gm of phenol may

be lethal to hujnans. Sometimes respiratory failure is also observed which results in

death (Clayton and Clayton, 1982). Chronic phenol poisoning is characterized by

systejnic disorders such as digestive disturbances, nervous system effects, possibly by

skin discoloration, eruptions and damage to the liver and kidneys (Parmeggiani,

1983). Concentrated phenol solutions are severely irritating to the human eye and

cause conjunctival swelling, the cornea becomes white and loses sensation. Loss of

vision has occurred in some cases. In addition to systemic effects, contact with the

solid or liquid can produce chemical burns. Erythema, edema, tissue necrosis and

gangrene have been reported (Hathaway et al., 1991).

52

2.1.9.3 On Environment:

Phenol is toxic to aquatic animals and in general it is most hazardous for fish.

Contamination of water by phenol could harm aquatic organisms and ecosystems. As

a volatile organic compound (VOC) it can be involved in reactions with other air

pollutants that form ground-level ozone, which can cause damage to crops and

materials.

2.1.10 Cancer Risk:

A study found a high risk of lung cancer among those exposed to phenol, although the

excess risk was stronger in short-term than in long-term workers. This result was not

replicated in three other studies which reported results on phenol and lung cancer,

although tv/o of them had very low statistical power. In the three studies reporting

associations with multiple cancer sites, a few elevated risks were reported, but not at

any cancer site in two or more studies. The pattern of results fails to demonstrate a

risk of cancer due to phenol exposure (IARC, 1999). The substance is a

suspected carcinogen.

53

2.2 BIODEGRADATION OF PHENOL BY AEROBIC GRANULATION

TECHNOLOGY:

The contamination of water supplies occurs with hazardous pollutants, the most

dangerous and problematic of pollutants are aromatic organic compounds (Atlas et

al., 1981 and Leahy et al., 1990). These compounds are resistant to degradation

because of the stability of aromatic rings present in them (Leahy et al., 1990). In

addition, many aromatic compounds are toxic at very low concentrations and many

are believed to be carcinogenic (Dipple et al., 1976; Sims et al., 1980 and National

Academy of Sciences, 1983).

Phenols are the major environmental pollutants, phenols and its derivatives are

present in the effluent of many industrial processes due to their wide spread use,

among them are oil refineries, petrochemical plants, coke conversion,

pharmaceuticals and resin industries. Veeresh et al., (2005) gives the concentration

and COD of many industrial phenolic wastewaters (Table 2.1). Phenol is also the

most frequently found pollutants in rivers and lands fill runoff water (Prasad and

Ellis, 1978). Phenol concentration of up tol0,000 mg 1"' has been reported in much

industrial wastewater (Fedorak and Hrudey, 1988).

Phenol is toxic at relatively low concentrations and is listed as priority pollutants by

the United States Environmental Protection Agency (Ghisalba, 1983). The toxicity of

phenol often results in the reduction of wastewater biotreatment even at relatively low

concentration (Hinteregger et al., 1992). The concentration of phenols as low as 1 mg

r' are toxic to some aquatic species (Brown et al., 1967) and at far lower

concentrations causes taste and odour problem in drinking water (Rittmann and

McCarty, 2001). Hence, the removal of phenol from wastewater is of obvious interest.

For the removal of aromatic compounds from the ground water various physical,

chemical and biological methods are used (Strier et al., 1980; Atlas et al., 1981;

Kobayashi et al.. 1982; National Academy of sciences, 1983 and Gils et al, 1986).

54

'%:5i=^5+cv ^v

Table 2.1: Characteristics of Major Phenolic Wastewater • . Vi^t.v ij^i-rt*^-^

TYPE OF WASTE PHENOLIC COMPOUNDS PHENOLIC COD

WATER CONCENTRATION (mg 1) (mg 1)

Coal gasification Phenol= 207 102.0

Lignite gasification 5500-7260

Coke plant ammonia still 620-1150

13,090-17,279

1476-2737

Petroleum refinery 6.42-88.03 15.3-210

Oil refinery 10-100

General petrochemical 50-600

Aircraft maintenance 200-400

Herbicide manufacturing 210

Plastic factory 600-200

Fiber board factory 150

Phenolic resin production 1600

55

Among these methods biological method is preferred if complete oxidation to carbon

dioxide and water is to be achieved. Physical and chemical methods have drawback

because in these methods intermediates are formed, that are even more toxic than the

original compounds (Strier et al., 1980 and Gils et al., 1986). For example, in the case

of phenol, bulk removal of phenol by solvent extraction, absorption, chemical

oxidation, incineration and other non biological treatment methods suffer from

serious drawback of high cost and formation of hazardous by products (Loh et al,

2000). Biological degradation is generally preferred due to the low cost and the

possibility of complete mineralization. Literature survey reveals that biodegradation

of phenols has already been a field of research by others (Table 2.2) although the

present method of biodegradation has the advantage of having higher organic load.

For many years phenol removal has been being carried out by activated sludge

system. This system is sensitive to high phenol loading rates and the fluctuations in

phenol loadings resulting in complete failure (Watanabe et al., 1999 and Kibret et a!.,

2000).

Phenol containing wastewater is difficult to treat because of substrate inhibition,

whereby microbial growth and concomitant biodegradation of phenols are hindered

by the toxicity exerted by the substrate itself

The inhibition difficulties associated with high strength phenolic wastewater can

overcome by strategies such as bioagumentation (Watanabe et al., 2002) and the other

one is cell immobilization (Keweloh et al., 1989). For example cells of Pseiidomonas

immobilized in polysulfones holiow fiber membrane were successful in degrading

phenol at concentration in excess of 500 mg 1', albeit at relatively slow rate (Loh et

al.. 2000). Aerobic granulation technology has been developed for treating a wide

variety of wastewaters (Peng et al., 1999; Moy et al., 2002; Lin et al, 2003 and Yang

et al., 2003). This technology has also been extensively investigated (Morgenroth et

al.. 1997: Beun et al.. 1999; Tay et al., 2001 and Liu and Tay 2004). Most studies

have either focused on granule cultivation through various substrates or treating

efficiency of \-arious granule processes (Arrojo et al., 2004; Kim et al., 2004;

McSwain et al.. 2004: Wang et al.. 2004 and Hu et al.. 2005).

56

Table 2.2: Organic Loading Rate of Phenols in Different Mediums

MEDIUM OBSERVATION REFERENCE

Mixed species Nakamura et al., 2000.

Maei/species Goudar et al., 2000.

Pseiidomonas Saet al., 2001.

piitida DSM 548

M/xeJ species Kryst et al., 2001.

Pseiidomonas The bioreactor showed phenol Gonzalez'et al., 2001.

putidfa degradation efficiencies higher

than 90%, even for a phenol

loading rate of 0.5 gm phenol

r' d"' (corresponding to 0.54

gm TOC r' d-').

A//x(?c/species Spence et al., 2001.

Rhodococcus DU i i f i Prieto et al., 2002.

Phenols completely

• ' biodegraded at a rate of 0.5 kg

phenol m' day' in the case of

WWl and partially

concentrations lower than 50

mg r' at 0.1 and 1.0 kg phenol

m"' d'' in the cases of WW2

57

and WWl, respectively.

Pseudomonas

pictonim (NCIM

2077)

Sheeja et al., 2002.

Mixed species Granules were successfully Jiang et al., 2002.

degraded 1.5 gm 1"' d'' phenol.

Pseudomonas Mrozic et al., 2002.

vesicularis and

Staphylococcus

sciuri

Ca-alginate W-17 can tolerate high Haleemet al., 2003.

Acinetobacter sp. concentrations of NH4^ -N (63

mg r') and N03~N (1000 mg

i-').

Chen etal., 2003.

Strain W-17

Comcunonas

testosterone ZD4-1

and Pseudomonas

arigenosa ZD4-3

Rhodococcus strain

(Nocardiaceae

family)

Alva etal , 2003.

58

Mixed species The total chemical oxygen Floreset al., 2003.

demand COD removal above

90% was achieved even at

organic loading rates as high as

7 kg COD m- d-'.

Mixed species

Mixed species

Jiang et al., 2004.

Mixed species

A maximum phenol- Lora et al., 2004.

biodegradation activity of 83

imol g"' VSS h-' was assessed

and the ICIOO values were 21.8

mmol dm" and 13.4 mmol d

m' for phenol and sulfide

respectively

Granules degraded 2.5 Tay et al., 2004.

kg phenol m~ d~'.

Fusarium

floccifenim

Mixed species

The phenol degraded below Mendonga et al., 2004.

200 mg r' detection limit.

Phenol concentration up to Tay et al., 2005.

500-750 mg T' generally not

inhibitory to UASB process.

59

Mixed species

Pseudomonas

putida

The new microbial consortium Prpich et al., 2005.

used within a TPPB was

capable of degrading high

concentrations of phenol (2000

mg r ' ) , with decreased lag

time (10 h) and increased

specific rate of phenol

degradation (0.71 gm phenol

g-' cell h).

The reactor performance at Pazarlioglu et al., 2005.

high phenol concentration for

the reactor tolerance was 1.25

gm r' phenol. The maximum

phenol degradation level of

99% was reached at a phenol

loading rate of 0.001-0.002 gm

1-'.

Pseiidomonas For pure culture P. Oborien et al., 2005.

oeniginosa NCIB aeruginosaio concentration

950, Pseiidomonas from 100 ppm (100 mg L"') to

fluorescence NCIB 250 and 500 ppm increased the

3756 lag phase 8 and 16 h,

respectively. Mixed cultures

had a lag phase only at

concentration of 500 mg 1"'.

Mixed species Gerrad et al.. 2006.

60

Mixed species Ladisalo et al.. 2006.

Propioniferrex PG- The culture of both strain Jiang et al., 2006.

02 and Comamonas degraded phenol at initial

sp. PG-08 concentration of 250 mg 1"'

faster than each strain

separately.

RWC-Cx\

Mixed species

RWC-Cx\ was not significantly Mailin et al., 2006.

influenced by acclimatization

and not affected by the highest

phenol concentration

employed, namely 1000 mg 1''.

Farooqi et al., 2007.

Mixed species Adav et al., 2007.

Actinobacillus sp. The maximum predicted Khleifat et al., 2007.

growth rate by this model is

0.37 h"' for NB and M4 media

obtained when the initial

concentration of phenol is 100

mgr'.

61

Klebsiella oxytoca K. oxytoca degrade phenol at Shawabkeh et al., 2007.

elevated phenol concentration

where 75% of initial phenol

concentration of 100 ppm will

degrade within 72 h. At phenol

concentration above 400 ppm,

the cells were unable to

degrade the substrate

efficiently.

M/xeJ species Nair et al., 2008.

Pseiidomonas Agarry etaj., 2008.

aeruginosa and

Pseudomonas

fluorescence

Ammonia-oxidizing Ghanavati et al., 2008.

bacteria and phenol-

degrading yeast

Mixed species The results showed that the Saravanan et al., 2008.

culture could degrade

phenol/m-cresol completely at

a maximum concentration of

600 mg r' and 400 mg f'

respectively.

Mixed species Wang et al., 2009.

Mixed species Lin cl al., 2009.

62

Corynebacterium sp These granules can degrade Ho et al., 2009.

DJl phenol at sufficient high rate

without severe inhibitory

effects up to phenol

concentration of 2000 mg F ' .

Candida Vermaet al., 2009.

tropicalis NCIM

3556

M/.Y£;<i species Saravanan et al., 2010.

C-14-1 {Nocardia Maet al., 2010.

sp.)

63

Aerobic granulation represents another cell immobilization technology that has

attracted resent research attention of researchers (Tay et al., 2001, 2002). Unlike

biofilm, aerobic granules are self-immobilized microbial consortia cultivated in SBR

without the inert support. The compact microbial structure can confer on aerobic

granules good settleability and high biodegradation capacity for toxic and recalcitrant

compounds (Jiang et al., 2002; Tay et al., 2005a and Yi et al, 2006). Aerobic granules

have been successfully cultivated in activated sludge fed with phenol as sole carbon

source (Jiang et al, 2002). With the kinetic data it is indicated that phenol degrading

aerobic granule have potential to treat wastewater with high phenol loading and also

possess high actively compact structure and good settleability. Because of phenol

toxicity there is a need to develop a biodegradation method with high organic load.

This study was undertaken for the biodegradation of phenol by microorganism

through sequencing batch reactor.

In present work the degradation of phenol aerobically in SBR has been achieved. The

main object of this study was to degrade phenol at a high loading rate of 3.9 kg m" d"'

and to study the aerobic degradation profile of phenol through an aerobic granulation

technology in a sequencing batch reactor (SBR).

2.2.1 Materials and Methods:

2.2.1.1 Reactor Set up and Operation:

Initially sludge was acclimatized in aeration tank (Fig 2.3) and then inoculated into

SBR. A column type sequencing aerobic sludge blanket reactor 5 cm diameter and

150 cm high (H/D 30) with a working volume 1.4 1 (total volume 1.5 1) and made of

transparent perfex glass was used. The reactor was operated at about 25 ±2* C. The

reactor was fed Viith phenol as a sole carbon source and was left open for the growth

of natural mixed population. A fine bubble aerator for supplying air at superficial air

velocity of 2.67 cm s"' was fitted at the bottom of column. Reactor was operated

sequentially in 4 hours cycle (5 minutes of influent filling. 228-230 minutes of

aeration. 2-30 minutes of settling and 5 minutes of effluent withdrawal). Fig 2.4 and

64

Fig 2.5 shows the SBR during aeration and settling. Fig 2.6 shows settled sludge in

SBR after aeration. To avoid excess loss of sludge, the reactor was initially operated

at high settling time of 30 minutes later the settling time was reduced to 10 minutes

and finally to 2 minutes. At the initial stage feeding time was kept high which was

decreased gradually as the operation proceeds. The reactor set-up included two

sampling port, arranged along the height of column type reactor, one at a distance of

70 cm and other at 30 cm from the bottom of reactor. The effluent was collected

through 70 cm port at a volumetric exchange ratio of 50% giving a hydraulic retention

time (HRT) of 8 hours. The HRT of 8 hours was kept constant throughout the study.

Port at 30 cm height was used for the collection of MLSS/MLVSS and periodical

sludge wasting. The operation proceeded with abiotic loss of phenol in SBR which

was negligible under identical operation condition.

2.2.1.2 Seed sludge and Wastewater:

The source of aerobic digested sludge with mixed liquor suspended solid (MLSS)

content of 3.5 gm 1"' and mixed liquor volatile suspended solid (MLVSS) content of

2.1 gm r' was taken from secondary clarifier of Star Paper Mill Saharanpur, India.

For the period of first 15 days, it was conditioned in an aeration tank where the

content was fed with 5 to 50 mg l ' phenol as the only source of carbon along with

macro (.Hang et al., 2002) and micronutrients (Moy et al. 2002). Composition of

nutrients is shown in Table 2.3. The acclimatization towards phenol was done at a

temperature of 25 ±2* C. After the period of acclimatization the aeration tank was fed

into sequencing batch reactor and used as starting culture for cultivating phenol

degrading aerobic granules. Synthetic wastewater containing 50 mg 1"' phenol in

mineral salt medium (nutrient solution) was given to SBR in batches of 4 hours each.

Phenol concentration was gradually increased to 650 mg 1"' in a stepwise process in

45 days period.

65

V

\

Fig 2.3: At the time of acclimatization wiir

phenol in activated sludge process.

Fig 2.4: SBR while aeration during a cycle

(with phenol).

66

v ^

K 1

T-" \

, * -

< « » * -t

1

#'

1 ' " ^ Fig 2.5: SBR at the time of settling (with '^'g 2.6: Settled sludge after aeration

phenol). (with phenol).

67

A slight alkaline pH (around 7.5) is necessary for proper aerobic granulation and

granulation cease to occur at pH> 8.5 as reported by (Hailei et al., 2006). Yang et al.,

(2008) reported that a slight low pH favors fungi dominating granules where as a

slight alkaline pH favors bacterial granules.

2.2.1.3 Wastewater Feed:

A stock solution of phenol was prepared having concentration of 10 gm 1"' (10,000 mg

r'). The desired concentrations of phenol for experiments were obtained by

proportionate dilution with double distilled water and fed into SBR. Micronutrient

were prepared at a 1000 times concentration. For optimal microbial growth 1 ml of

each was added to the feed solution. Influent wastewater pH was adjusted to around

7.5 by adding NaHC03 (Moy et al., 2002 and Jiang et al., 2002).

2.2.1.4 Analytical Methods:

Measurement of pH, suspended solids, mixed liquor suspended solids (MLSS), mixed

liquor volatile suspended solids (MLVSS), chemical oxygen demand (COD) was

conducted in accordance with the standard methods (APHA, 1998). The phenol

concentrations were determined spectrophotometrically using the absorbance values

at 500 nm with a UV/VIS spectrophotometer (GENESYS 20, Thermospectronic)

according to APHA, (1995). Percent COD removal and phenol removal was

determined using the following relations:

Influent COD-Effluent COD . , „ „ % COD removal = * 100

Influent COD

Influent Phenol-Effluent Phenol , , „„ % Phenol removal = " ^ ] 00

Influent Phenol

Growth of the cell was measured by noting optical density (O.D.600) as discussed by

Ziagova et al., (2007). O.D.goo was calculated using UV/VlS spectrophotometer

(GENESYS, Thermospctronic) at 600 nm A|„ax taking double distilled water as blank.

68

Table 2.3: Composition of Nutrients (phenol)

NUTRIENTS

Macro Nutrients

KH2PO4

K2HPO4

MgS04.7H20

NH4CI

Micro Nutrients

H3BO3

ZnCU

CuCl,

MnS04.H20

M07O24.4H2O

AICI3

C0CI2.6H2O

NiCl

AM'

1.35

1.65

0.13

0.20

0.05

0.05

0.03

0.05

0.05

0.05

0.05

0.05

AMOUNT REQUIRED (gm l')

69

Specific growth rate was estimated from the slope of exponential rise phase of growth

curve (O.D.goocurve of SBR) as specified by Ziagova et al., (2007).

For calculating specific degradation rate (SDR) and degradation rate (DR) from the

concentration following relations were used:

, , Influent Phenol-Effluent Phenol (mg) SDR (mg phenol gm VSS"' hr"') = TTrrTr—T"" 'T'.—^^

^^ ^ ^ MLVSS (gm) Time (hr)

, , Influent Phenol Cone-Effluent Phenol Cone, (mg/1) DR (mg r' hr-') = -— ^ - ^ ^

^ ^ ' Time(hr)

Morphology and surface structure of granules were observed qualitatively with a

scanning electron microscope (STEREO SCAN 360) according to method discussed

by Ivanov et al., (2006). Granules were prepared for SEM image by washing vv ith a

phosphate buffer and fixing with 2% glutaraidehyde overnight at 4 C. Fixed granules

were washed with 0.10 M sodium cacodylate buffer and dehydrated by successive

passages through 25, 50, 75, 80, 90, 95 and 100% ethanol and dried with a CO2

Critical Point Dryer.

2.2.2 Results and Discussion:

Phenols are most common compounds, distributed in waste discharge of pulp and

paper industries. The micro organism present in such waste are already acclimatized

for phenol and its derivatives through natural selection. It was due to this reason we

selected the aerobic sludge from Star Paper Mill Saharanpur, India. This already

acclimatized sludge reduces conditioning period in our case. Initially sludge was

acclimatized for about 15 days to allow biomass to adapt phenol, for getting more

specific microbes for phenol degradation.

70

2.2.2.1 Biomass Concentration (MLSS and MLVSS):

The initial sludge has MLSS concentration of 3.5 gm 1"' and MLVSS of 2.1 gm 1"'

which increases to 5.2 gm 1"' and 3.08 gm 1"' respectively after conditioning

(acclimatization) period. Then the sludge was inoculated in to SBR for the cultivation

of aerobic granules. Aerobic granules developed along with amorphous floes were

first observed after 10 days of inoculation. However, MLSS concentration to 3.5 gm

r' and MLVSS to 2.03 gm 1"' due to reduction in settling time from 30 minutes to 10

minutes (on day 13*). The biomass with small settling velocity had been washed out

of reactor. Small granules grew rapidly in subsequent week, while more floe like

sludge washed out from reactor gradually. Further, the MLSS and MLVSS

concentration increases till 5.6 gm 1'' and 3.58 gm f' and again shows a sharp

decrease, due to decrease in settling velocity from 10 to 2 minutes on 23" day. The

granules become dominant form of biomass as evident from gradual increase in

biomass concentration beyond day 24. MLSS and MLVSS concentration finally

shows an upward trend and stabilizes at 7.3 gm 1"' and 4.72 gm f' (Fig 2.7).

2.2.2.2 Shape and Size of Granule:

Stable granules were obtained after 45 days of SBR operation. High degradation

efficiency of granules is due to their compact structure and not effected by shape of

granules (Khan et al., 2009). Granules have a high resistant to toxic xenobiotic due to

their compact structure (Glancer et ai., 1994; .Jiang et a!., 2002; Tay et al., 2005b and

Vlami et al.. 2005). Fig 2.8 a shows microscopic image of sludge after one week of

inoculation. Fig 2.8 b and c shows SEM image of granules at 100 at 15,000

magnifications. The diameter of granules was about 1-2 mm shows close pack

structure with evenly distributed channels which facilitates movement of water inside

the granules. The degradation capability of aerobic granules depends mainly on their

compactness.

71

Days

Fig 2.7: Variation of MLSS and MLVSS concentration of phenol during its

degradation.

(a)

Fig 2.8 a: Microscopic image of sludge (after one week of inoculation)

72

(b)

(c)

Fig 2.8 b«& c: SEM image of mature granules at 100 and 15,000 magnifications.

73

2.2.2.3 Microbial Density of SBR and Effluent:

Microbial density of effluent and SBR is measured in terms of optical density

(O.D.6oo)- Fig 2.9 illustrates that in the beginning O.D.goo of effluent was 1.67 and

O.D.600 of the SBR was 1.82. Due to reduced settling time on 13"' day (30 to 10

minutes) and 23* day (10 to 2 minutes) O.D.eoo of effluent increases and O.D.soo of

SBR decreases. From day 1 ' to 12* day when the settling time was 30 minutes,

O.D.600 of effluent decreases from 1.67 to 0.9 and the SBR O.D. oo increases from

1.82 to 2.17, but on day 13* when the settling time was reduced to 10 minutes the

O.D.600 of effluent increases to 1.05 and then reduces gradually. However, O.D.600 of

SBR decreases to 1.83 on day 13* and then increases gradually up to day 22"'' with

same settling time. There is again an increase in effluent O.D.600 value to 0.81 and

decrease in the value of O.D.600 of SBR to 1.96 on day 23''' due to reduction in settling

time to 2 minutes. Thereafter, values of O.D.600 of effluent decreases and got

stabilized at 0.15, meanwhile SBR value increases for the rest of the cycle up to day

40 and then stabilizes at 2.64. Specific growth rate of the system was estimated to be

0.019 d"'. Reduction in settling time leads to biomass washout in effluent which is a

pre-requisite criteria for aerobic granulation. Since granules are strong and compact in

structure, their settling is faster due to gravity. Thus, the effectiveness of the chosen

operating strategy was reflected by decrease in O.D600 of the effluent.

2.2.2.4 Phenol Removal Profile:

The degradation profile shows that initially the degradation of phenol was high later

on degradation rate decreases till the end of cycle (Fig 2.10). This is because iniUally

microorganisms has large amount of phenol, so they degrade it faster. This means

degradation rate is directly proportional to substrate to a certain level. During the first

60 minutes of 240 minutes cycle (with 50% volumetric exchange giving 8 hours

HRT) sufficient amount of phenol is removed but in next 180 minutes its

concentration reduces to as low as 8 mg f'. Fig 2.10 also shows that the concentration

of 50 and 100 mg f of phenol decreases to below detection limit in just 150 minutes

but high concentration of phenol (200,400. 650 mg I'') took around 220-230 minutes

74

for complete removal. Just after addition of influent, a sharp decrease in phenol

concentration was observed (as shown in Fig 2.10 first 60 minutes), this is due to

dilution i.e. when we add influent containing phenol in SBR, then due to presence of

wastewater in SBR, phenol concentration decreases due to dilution. Phenol

degradation rate at lowest (50 mg 1"') and highest (650 mg 1'') concentration treated in

this study were 11.29 mg 1"' hr'' and 153.24 mg 1"' lir"' with corresponding removal

efficiency of 90.36% and 94.3% respecfively.

Specific degradation rate shows the growing order which gets stabilized at 650 mg f'

32.46 mg phenol gm VSS"' hr"'. Afterwards it was decreased because of substrate

inhibition (Kargi and Eker 2005).

2.2.2.5 COD Removal Profile:

Similar is the case with COD removal. The removal rate of COD was high in the

beginning later on it goes on decreasing. The sharp decrease in COD was observed

because of dilution just after addition of influent in the SBR. This value of COD

further decreases to give COD concentration below 200 mg 1"' at highest load of

phenol was by microbial acfion (Fig 2.11). Removal efficiency was 95.61% and

97.39%) respectively at lowest and highest loadings.

2.2.2.6 Variation of COD along with Percent COD Removal Efficiency during

Biodegradation of Phenol:

Fig 2.12 reflects variation of COD of influent and effluent along with percent COD

removal efficiency. Initially the influent COD was not very high (570 mg 1'') still the

effluent COD fluctuate 215 mg f' along with low removal efficiency 62.28%),

because of lack of granulation. As soon as the loose microbial floes changes into thick

granules, effluent COD decreased below 200 mg f' even when the influent COD was

quite high 7418 mg 1''. At this stage removal efficiency was 97.39%).

75

2.5 -

2 . 0 -

• * • • »

'aft

C

"si _u

1 I O ­

CS -

0 0 -

Effluent L V SBR

~ " „ -;.•

\

•i'

"- .r-1 • 1 1 ' 1

.. < ? '•" '^^ • ' ' •

,

• - - -

10 20 30

Davs

40 50

Fig 2.9: Optical Density (600nm) of effluent and SBR variation with time.

150

Time (min)

r 250

Fig 2.10: Concentration profile of phenol during single cycle.

76

k\y"^ •* -

''-/•.;T^83T-o ;

y>1'n . J n i ^ ^ '

8000 -

7 0 0 0 -

6 0 0 0 -

'•^5000 -

g ^4000 -S3

> O g 3000 -u Q:

O 2000 -

o u 1000 -

0 -

<

,:

#

t i,^};

---• . __\_ ' __ f #

1 • ' 1 • ' 1 T - 1

^ 50 mg r'

100 mg I ' 200 mg 1'

400 mg 1' o 650 mg r'

- - • • . ^ ^ . . . . . >

1 ' 1

50 100

Time (min)

150 200 250

Fig 2.11: COD removal profile of phenol during single cycle operation.

9000

8000

7000

6000

'^- 5000 -M E ^ 4000 Q O U 3000

2000

1000-

0

— •» — In f luen t

— — Eff luent

' % R e m o v a l

:**_\: / • , y \ / \ / \ / \ / \ / \ / \ / " . : / ^ y -

0 • ~ r -10 20

I 30 40 50

Davs

Fig 2.12: Percent COD removal efficiency along with effluent and influent COD.

77

2,2.2.7 Phenol Removal along with Percent Removal Efficiency:

Fig 2.13 shows that initially 50 mg 1"' of phenol was fed into SBR and effluent

concentration was 27.56 mg f' and low removal efficiency of 47%. The behavior

remains same for few days. However, in the subsequent week when floes

transforming into granules due to wash out of poor settling floes at a selected

pressure, system becoming stable and removal efficiency increases. Thereafter the

influent concentration was increased stepwise from 50 to 650 mg 1"' but the system

sustaining the ill effects of the rising phenol concentration. As the granules become

stable and compact, removal efficiency increases, removal efficiency was 94.35%

achieved at the end of the operation with the effluent phenol concentration of around

37 mg r' and influent concentration around 656 mg 1" .

2.2.3 Conclusion:

Preconditioned sludge takes less acclimatization duration due to the presence of

already acclimatized microbes from natural selection. This study demonstrates that

well settling granules for phenol degradation can be cultivated in SBR which can

tolerate fluctuation in organic load of 570 mg COD f' to 7500 mg COD 1"', maximum

phenol concentration of 650 mg f' is treated which is equal to 3.9 kg m" d''. Optical

density O.D.goo of effluent stabilized at 0.15 which minimizes the loss of biomass in

effluent and same microbes can be used over a long period. Optical density (O.D. 6oo)

of SBR stabilized at 2.64 which increase biomass during the SBR operation cycle.

The specific growth rate was found to be 0.019 d '. MLSS and MLVSS were

stabilized at 7.3 and 4.72 gm f'. The graph of concentration of effluent and influent

Vs percent phenol removal efficiency gives 94.3% and COD removal efficiency was

97.39%. The degradafion rate increases with dme during a single cycle of operation.

Degradadon profile illustrates that during first hour of cycle removal of phenol and

COD were high but later on becomes low. In single cycle degradation rate of phenol

was 11.29 mg 1"' hr"' and 153.24 mg !"' hf' at 50 mg 1"' and at 650 mg f'

concentration (highest load).

78

Removal efficiency of phenol at lower and higher concentration was 90.36% and

94.3% respectively. Removal efficiency of COD at 50 and 650 mg l ' was 95.61% and

97.39% respectively. At maximum loading removal efficiency was high than

minimum loading, this is due to the fact that initially there was no formation of

compact granules so the concentration of effluent was high and influent was low in

the beginning. But afterwards the concentration of effluent becomes lower however

the concentration of influent is higher as the microbes start degrading it effectively

because of compact granules. The specific degradation rate was 32.46 mg phenol gm

VSS-' hr"'.

This process appears very useful for treating wastewater containing phenol and

advantageous as the same biomass can be used for a long period of time. This process

can withstand high organic load as well as the fluctuations in organic load so it can be

used for treating municipal and industrial waste both.

79

900

800

CD S 600

c •2 500

U

a 400 u e o U 300

S 200

100 -

"

/ • . /

1 —

1 ' 1 ' I

/ /

/ y

/ * /

— —

/ '--/

1 ' 1 • 1

1 1 1

/ /

/

— Influent — Effluent

% Removal -

• r • 1 10 20 30 40

Davs

50

Fig 2.13: Percent phenol removal Vs effluent and influent concentration.

80

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91

CHAPTER 3

AEROBIC GRANULATION FOR PARA

NITRO PHENOL BIODEGRADATION IN A

SEQUENCING BATCH REACTOR

Aerobic Granulation for Para Nitro Phenol Biodegradation in a

Sequencing Batch Reactor

Para Nitro Phenol (PNP) is a xenobiotic found in mainly dye, explosive and pesticides

industries effluent. It should be treated aerobically because under metanogenic

conditions (anaerobic degradation) PNP produce much more toxic amino derivatives.

Aerobic granules tolerate and degrade PNP at levels that were known to cause

breakdown of activated sludge process. Activated sludge process might not be

suitable with a higher PNP concentrations and fluctuations in PNP case. The

development of aerobic PNP granules was the conditioning of activated sludge seed

for biomass that has improved settleability and higher PNP degradation activity.

Microorganisms are capable to degrade PNP (Donlon et al., 1996). PNP has a nitro

group which has electron withdrawing nature, this factor makes the PNP recalcitrant

to oxidative cleavage of aromatic ring in aerobic process (Melgoza et al., 2001). The

presence of nitro group makes it more challenging to microbial degradation.

A brief introduction of PNP includes properties, uses and health hazard information

as follows.

3.1 PARA NITRO PHENOL:

Para Nitro Phenol (PNP) is a phenolic compound that has a nitro group at the opposite

position of hydroxy! group on the benzene ring.

PNP is used as a chemical intermediate in the production of methyl and ethyl

parathion, N-acetyl-p-aminophenol dyestuffs and leather preservatives (HSDB, 1991).

PNP is a federal hazardous air pollutant and was identified as a toxic air contaminant

in April 1993 under AB 2728. The annual production of PNP in the U.S. from 1989 to

1994 is about 22 to 25 million pounds (HSDB. 1999).

No information about the natural occurrence of PNP was found in the available

literature. It has been detected in the exhaust gas of motor vehicles and as an impurity

in the parathion formulation Thiophos (Howard. 1990).

92

3.1.1 Structure:

Any compound derived from phenol by the replacement of one of its ring hydrogen

atoms by the nitro group is PNP (NO2C6H4OH). Molecular structure of PNP and

resonance structure of PNP anion are shown in Fig 3.1 and Fig 3.2.

3.1.2 Appearance:

PNP is a white to yellowish powder. It shows two polymorphs in crystalline state. The

a-form is colourless pillars, unstable at room temperature and stable towards sunlight.

The P-form is yellow pillars, stable at room temperature and gradually turns red upon

irradiation to sunlight. Usually PNP exists as a mixture of these two forms.

Solution of PNP alone appears colourless or pale yellow, whereas its phenolic salts

tend to develop a bright yellow colour. This colour changing property makes PNP

useful as a pH indicator.

3.1.3 Odour: Odourless but a sweet then burning taste (HSDB, 1991).

3.1.4 Physical Properties:

• Molecular Weight: 139.11.

• Boiling Point: 279" C.

• Melting Point: 113-114* ^ C, It sublimes and is slightly volatile with steam

(HSDB. 1991). If mixed with diethyl phosphite may explode when heated. It is

decomposed upon heating and emits toxic fumes of nitrogen oxides.

• Density/Specific Gravity: 1.27 at 120/4" C (water). ,

• Density: 1.5 gm cm'"\

• Vapour Pressure: 0.001 mm Hg at 25" C.

• Solubilit)': Alcohol, chloroform, acetone, pyrimidine, toluene, hot benzene, hot

water, ether, fixed alkali hydroxide solutions and carbonates.

• . Water Solubility: 1.6g/l 00 ml (25" C).

• pH: 5.6 (colourless) to 7.6 (yellow).

93

OH

NO:

FigS.l: Structure of PNP

/ \

o- o-

Fig 3.2: Resonance structure of the PNP anion

94

Dissociation Constant (pKa): 7.08 at 22* C.

3.1.5 Chemical Properties:

3.1.5.1 Reactivity: Violent chemical change possible.

• Potentially Hazardous Incompatibilities: Mixtures with potassium hydroxide

are explosive.

• Water Reactions: Soluble in hot water and denser than water

3.1.5.2 Flammability: Must be preheated to burn.

• Flashpoint: 169° C.

• Auto ignition Temperature: 490° C.

• Fire Hazard: Toxic oxides of nitrogen and fumes of unburned material may

form in fires.

• Behaviour in Fire: Decomposes violently at 279° C and will burn even in

absence ofair(USCG, 1999).

3.1.5.3 Decomposition: Decomposes exothermally, emits toxic fumes of oxides of

nitrogen (Lewis 3" ed., 1993 and Sax, 1989). Decomposes violently at 11 f C

and will burn even in absence of air (USCG, 1999). Solid mixtures of the

nitrophenol and potassium hydroxide (1:1.5 mol) readily deflagrate

(Bretherick 5"'Ed., 1995).

3.1.5.4 Explosion: May explode on heafing. The substance decomposes on heating

producing toxic fumes including nitrogen oxides. Mixtures with potassium

hydroxide are explosive.

3.1.5.5Health Hazard: PNP is extremely hazardous.

95

3.1.6 Uses:

• PNP has been used as a model substrate for cytochrome P450 2Ei, although the

major urinary metabolite is its glucuronide conjugate.

• PNP is both a breakdown product and a metabolite of parathion and its

derivatives.

• PNP is used as a raw material in the synthesis of dyes, pharmaceuticals,

pesticides, polymers, phosphorous inorganic insecticides, explosives, solvent and

pigments.

• In the synthesis of paracetamol, PNP is used as an intermediate. It is reduced to 4-

aminophenol, then acetylated with acetic anhydride.

• For the preparation of phenetidine and acetophenetidine, indicator and raw

material of fungicides, PNP is used as the precursor. Bioaccumulation of this

compound is considered to occur rarely.

• In the peptide synthesis, carboxylate ester derivatives of PNP may serve as

activated components for a construction of amide moieties.

3.1.7 Health Hazard Information:

The main source of information for the toxicity of PNP is the Agency for Toxic

Substances and Disease Registry's (ATSDR's) toxicological profile for nitrophenols.

Other secondary sources include the Hazardous Substances Data Bank (HSDB). a

database of summaries of peer-reviewed literature, and the Registry of Toxic Effects

of Chemical Substances (RTECS), a database of toxic effects that are not peer

reviewed.

3.1.8 Route of Exposure: The substance enters into the body by inhalation, through

the skin and by ingestion (U.S. EPA, 1994a).

3.1.9 Health Effects of PNP:

PNP affects the eyes, skin, respiratory tract, blood and causes the inflammation.

96

3.1.9.1 On Animals:

• PNP from inhalation exposure in rats reported effects on respiratory system, an

increase in methemoglobin and property on the liver and corneal opacity

(ASTDR, 1990).

• The LD50 test in rats and mice, have shown PNP to have high toxicity from oral

and dermal exposure (U. S. RTECS, 1993).

• PNP from dermal exposure reported no effects on the respiratory, cardiovascular,

gastrointestinal, muscular, immune, central nervous systems, liver and kidney.

The only effects noted were dermal irritation consisting of erythema, scaling,

scabbing and cracking of the skin (ASTDR, 1990).

• One animal study reported no histological alterations in the testes and

epididymides in mice exposed to PNP by inhalation, while in another study no

changes were observed in the reproductive index of pregnant mice given PNP by

gavage (placing the chemical experimentally in the stomach) (ASTDR, 1990).

3.1.9.2 On Humans:

• Acute (short-term) inhalation or ingestion of PNP in humans causes headache,

drowsiness, nausea and cyanosis (blue colour in lips, ears and fingernails).

Contact with the eyes causes irritation (U. S. HSDB, 1993).

• No information is available on the chronic (long-term) effects of PNP in humans

or animals from inhalation or oral exposure (ASTDR, 1990).

• EPA has determined that there are inadequate data for the establishment of RfC

(Reference Concentration) for PNP.

• The RtD (Reference Dose) for PNP is under review by EPA (U. S. IRIS, 1993).

• No information is available on the reproductive or developmental effects of PNP

in humans (ASTDR. 1990).

97

3.1.9.3 On Environment:

PNP is low to moderately toxic to birds and aquatic animals. Wild animals or plants

are not likely to be exposed to PNP, since it is not applied outside of a factory. Thus

PNP is not expected to pose a risk to non-target organisms (U.S. EPA, 1998).

3.1,10 Cancer Risk:

The International Agency for Research on Cancer and the U.S. EPA has not classified

PNP for potential carcinogenicity (lARC, 1987a and U.S. EPA, 1994a).

98

3.2 BIODEGRADATION OF PARA NITRO PHENOL BY AEROBIC

GRANULAR SLUDGE PROCESS:

Nitrogen containing aromatic compounds has industrial importance, these compounds

are used as a raw material in the synthesis of dyes, pharmaceuticals, pesticides,

polymers, phosphorous organic insecticides, explosives, solvent and pigments

(ATSDR 1992, Podeh et al., 1995; Dieckmann and Gray 1996; Kiwi et al., 1996;

Podeh et al., 1996; Uberoi et al, 1997; Ma et al., 2000 and Karim et al., 2001). These

compounds show more resistance than the unsubstituted compounds to degradation

because of the presence of the nitro group. Several research groups have been worked

on the biodegradation of nitrophenols (Simpson et al., 1953; Raymond et al., 1971;

Munnecke et al., 1974; Sudhakar et al., 1976; Spain et al., 1979; Gunderson et al.,

1984; Nyholm et al., 1984; Spain et al., 1984 and Zeyer et ai., 1984). Nitroaromatic

compounds pose a danger to human health, because of their high mutagenicity and

carcinogenicity (Schackmann, 1992).

Out of these nitroaromatic compounds, PNP is one of them. In terms of quantities

used and potential environmental impacts, PNP is one of the most important nitro

aromatic compounds (Podeh et al., 1995). The presence of nitro group makes PNP

more toxic so it is more challenging to microbial degradation. Through the photo

chemical reaction between benzene and nitrogen monoxide, PNP may be produced in

the atmosphere and has been detected in rain water in .lapan (EPA, 1980 and Karim et

al.. 2001). PNP is formed by hydrolysis of pesticides and herbicide and thereby

released into the subsurface and contaminate ground water resources (Labana et al..

2005). The PNP has been listed as a priority pollutant by United State Environmental

Protection Agency (U. S. EPA). EPA recommended restricting PNP concentration in

natural water below 10 ).ig f' (EPA, 1976) and the maximum limit is 20 |.ig P' (Chen

and Ra}-, 1998). The average PNP concentration should not exceed than 162 \.[g p'

(EPA, 1988). PNP being so carcinogenic, mutagenic and toxic causes significant

health risk therefore these limits has been decided. PNP causes blood disorder in

animals. Methemoglobiii formation, liver and kidney damage, anaemia, systematic

99

poisoning, skin and eye irritation can-occur by acute exposure of PNP (ATSDR, 1992

and HSDB, 1999). The removal of PNP from industrial wastewaters and ground

waters is necessary because of the above hazardous effects.

Nitroaromatic compounds are toxic to the microorganisms and they are resistant to a

complete biodegradation (Spain, 1995). An anaerobic degradation process is also used

to treat PNP containing wastewater (Tseng et al., 1995; Danlon et al., 1996; Karim et

al., 2001; Melgoza et al., 200land Bhatti et al., 2002). The anaerobic degradation

under methanogenic condition is a slow process in which PNP form much more toxic

amino derivatives (Gorontzy et al., 1993 and 0 'Connor and Young 1993).By

granular sludge in UASB, the anaerobic biotransformation and degradation was

investigated (Danlon et al.. 1996). When an anaerobic biological fluidized bed is used

to treat a synthetic wastewater containing nitroaromatic compounds result shows that

PNP is most toxic (Treng and Yang, 1995).

Under aerobic conditions, PNP is mineralized by microorganisms, so aerobic

treatment can be a viable alternative for PNP removal. PNP contaminated wastewater

should be treated aerobically. The higher amount of PNP degrading bacteria is found

in immobilized system, hence achieving higher PNP degradation activity and higher

tolerance to PNP toxicity (Ray et al., 1999 and Xing et al., 1999). Combined process

is such as anaerobic or aerobic is a viable alternative for the treatment of PNP that are

difficult to treat by traditional process. The removal of PNP has been possible by

using sequential anaerobic or aerobic process (Zitomer and Speece. 1993 and Table

3.1).

Aerobic granulation represents a cell immobilization technology that has attracted

recent research attention (Tay et al., 2001, 2002). Without the inert support for

biofilm attachment, aerobic granules are microbial aggregate cultivated in SBR.

Immobilized systems were able to retain higher amount of PNP degrading bacteria,

achieving higher PNP degradation activity and higher tolerance to PNP toxicity. PNP

is toxic so there is a need to develop a biodegradation method with high organic load.

100

TaWe 3.1: Organic Loading Rate of PNP in Different Mediums

MEDIUM OBSERVATION REFERENCE

Noairdioidcs sp. NSP41 The initial concentration of

phenol was increased from 100

to 400 mg r' the specific PNP

degradation rate at the 200 mg f'

was decreased from 0.028 to

0.021 h-'.

Cho et al., 2000.

Mixed species Flemming et al., 2000.

Ralstonia sp. The maximum rates of PNP

SJ98^ Artlvohacter degradation (F„„J 11.76, 7.81,

protophormiae RKJIOO and 3.84 |.miol PNP degraded

, and Biirkholderia min"' mg'' dry biomass,

cepacia RKJIOO respectively.

Bhushan et al , 2000.

Mixed species Qiao etal.. 2000.

Mixed species

Mixed species

Mixed species

Global efficiencies of PNP

mineralization of 98% were

obtained. The reaction rates

were 16 mg PNP !"'h"'.

AMio of > 1 in terms of

glucose to PNP could achieve

Beun etal.. 2001.

Melgoza etal.. 2001

Swaminathan et al..

2002.

101

Pseudomonas sp.

YTK17

and Rhodococcus

opacus YTK32

Mixed species

Mixed species

Rhodococcus sp.

Mixed species

Mixed species

Rhodococcus

opacus ^kOXQX

90% PNP degradation. However

the total organic carbon (TOC)

removals were 76.3%.

Shinozakaki et al., 2002.

Bhatti et al., 2002.

Tomei et al., 2003.

Takeo et al., 2003.

The maximum removal rate was Tomei et al., 2004.

in the range of 3.3-8.4

mg 4NP mg VSS"' d^').

Yang et al., 2004.

Kitagawa et al., 2004.

A//'.Yt'c/species Nitrogen loading and efficiency Arrojo ct al.. 2004.

were 0.7 N COD r'd"'and 70%.

Rhodococcus

wratislaviensis

This indigenous bacterial strain Gemini et al., 2005.

degrades 0.36 and 0.72 mM PNP

in 34 and 56 h, respectively.

Pseudomonas sp. Qureshi et al., 2005.

102

Pseudomonas

putida JS444

Mixed species

Lei et al, 2005.

Mixed species

Mixed species

Mixed species

Mixed species

The total p-NP and COD Sponza et al., 2005.

removal efficiencies were found

as 96 and 97% at loading rate of

3.85 gm p-NP m"^d-'.

The aerobic granules were Yi et al., 2006.

cultivated at a PNP loading rate

of 0.6 kg m" d"', with glucose to

boost the growth of PNP-

degrading biomass.

Qin et al., 2006.

In different nitro aromatics 0.3 Liu et al., 2006.

kg 4-NP m' -d"' removed.

Tomei et al., 2006.

Ochrobactriim sp. B2 The results shov\ed B2 was

capable of degrading diverse

aromatic compounds, but amino-

substituted benzene compounds,

at a concentration up to 100 mg

r' in 4 days.

Zhong et al., 2007.

103

Pseudomonas

pseudomallei EBN-10

Mixed species

EBN-12 mutant strain could Rehman et al., 2007.

completely mineralize PNP (100

mg r') on the minimal media in

24 h while, the parent strain

utilized only 6%.

SVI is less than 100 ml gm"'. Pratt et al., 2007.

Mixed species

Mixed species

Arthrobacter sp.

Microbes were degraded 0.3 kg Liu et al., 2007.

PNPm-^d''.

Zhang et al., 2008

The bacterium could tolerate Li et al., 2008.

concentrations of PNP up to 600

mg r ' , and degradation of PNP

was achieved within 120 h of

incubation.

Mixed species Hofstetter et a!.. 2008

Pseudomonas sp. strain

NyZ402

Mixed species

Wei et al.. 2009.

At the end of acclimation, higher ^^^^^^a et al., 2009.

values of growth yield (0.39 < Y

(PNP) < 0.63 mg COD(X) mg

COD PNP"' and maximum

growth rate 1.09 < |.i,„,,, PNP <

2.01 d"' were obtained.

Mixed species Complete decomposition of PNP Zeinab et al.. 2009.

104

13 mg r' by R. eurtopha was

occurred in 20 hours at 30 C.

Escherichia de La et al., 2010.

co//DH10band

two Psendomonas

pulida strains

105

The main object of this work was to degrade PNP at a high loading rate of 2.1 kg m"

d"' and to study the aerobic degradation profile of PNP through aerobic granulation

technology in a sequencing batch reactor (SBR).

3.2.1 Materials and Methods:

3.2.1.1 Reactor Set up and Operation:

Initially sludge acclimatized in aeration tank after 30 days inoculated in SBR (Fig

3.3). A column type sequencing aerobic sludge blanket reactor (5 cm diameter and

150 cm high) with working volume 1.4 1. (total volume 1.5 1) made of transparent

perfex glass was used. It was operated at about 30 ±\^ C. The reactor was fed with

PNP as a sole carbon source and was left open for the growth of natural mixed

population for 60 days period. Fine bubble aerator was fitted at the bottom of column

for supplying air at superficial air velocity of 2.67 cm s"'. Reactor was operated

sequentially in 4 hours cycle (5 minutes of influent filling, 200-228 minutes of

aeration, 2-30 minutes of settling and 5 minutes of effluent withdrawal). Fig 3.4 and

Fig 3.5 shows the SBR during aeration and settling and Fig 3.6 shows settled sludge

in SBR after aeration. To avoid excess loss of sludge, the reactor was initially

operated at a high settling time of 30 minutes. Later the settling time was reduced to

10 minutes and finally to 2 minutes. The reactor set-up included two sampling ports,

arranged along the height of column type reactor, one at a distance of 70 cm and other

at 30 cm from the bottom of the reactor. The effluent was collected through 70 cm

port at a volumetric exchange ratio of 50% giving a hydraulic retention time (HRT) of

8 hours. The HRT of 8 hours was kept constant during the analysis. The use of 30 cm

high port was for the collection of sample for MLSS/MLVSS determination and

periodical sludge wasting. The operation proceeded with abiotic losses of PNP in

SBR which were negligible (less than 5 %) under identical operating condition.

106

Fig 3.3: At the time of acclimatization witr.

PNP in activated sludge process.

Fig 3.5: SBR while aeration during a

cycle (with PNP).

107

Fig 3.5: SBR at the time of settling (with Fig 3.6: Settled sludge after aerati(Mi

PNP). (with PNP).

108

3.2.1.2 Seed Sludge and Wastewater:

The source of aerobic digested sludge with mixed liquor suspended sohd (MLSS)

content of 2.2 gm 1"' and mixed hquor volatile suspended solid (MLVSS) content of

0.32 gm r' were taken from secondary clarifier of Star Paper Mill Saharanpur, India.

For the period of first 30 days the content was fed with PNP as an only source of

carbon in mineral salt medium (nutrient solution) as specified by Yi et al , 2006 and

Moy et al., 2002. The acclimatization towards PNP was done at a temperature of 30

± f C. During acclimatization period only 5 mg l ' PNP was added to the reaction

mixture and then its concentration was gradually increased to 40 mg 1"'. After the

period of acclimatization the aeration tank was fed into SBR and started with

synthetic wastewater containing nutrients and 40 mg f' PNP which was gradually

increased to 350 mg 1"' in a stepwise procedure. The whole process was performed at

30 ±1° C temperature. Influent wastewater pH was adjusted to around 7.5 by adding

NaHCOs.

A slight alkaline pH (around 7.5) is necessary for proper aerobic granulation and

granulation cease to occur at pH> 8.5 as reported by (Hailei et al., 2006). Yang et al,

(2008) reported that a slight low pH favors fungi dominating granules where as a

slight alkaline pH favors bacterial granules. Temperature change also affects the

reactor performance up to a certain degree. Song et al, (2009) reported that 30' C is

optimum temperature for matured granule cultivation.

3.2.1.3 Wastewater Feed:

A stock solution of PNP was prepared having concentration of 4 gm 1" (4,000 mg 1"')

in 0.1 N NaOH. The desired concentrations of PNP for experiments were obtained by

proportionate dilution with double distilled water and fed into SBR along with

macronutrient given by Yi et al., 2006 (Table 3.1). Stock solution of micronutrients

(Table 3.2) were prepared at a 1000 times concentration (Moy et al , 2002). For

optimal microbial growth 1 ml of stock solution was added to every batch of the feed

solution.

109

Table 3.2: Composition of Macronutrients (PNP)

MACRO NUTRIENTS AMOUNT REQUIRED (mg 1')

KH2PO4 200

Na2HP04 650

MgS04.7H20 25

FeS04.7H20 20

CaCl2H20 30

Table 3.3: Composition of Micronutrients (PNP)

MICRO NUTRIENTS AMOUNT REQUIRED (gm 1^

H3BO3 0!05

ZnCU 0.05

CuCl. 0.03

MnS04.H20 0.05

M07O24.4H2O 0.05

AICI3 0.05

C0CI2.6H2O 0.05

NiCl 0.05

110

3.2.1.4 Analytical Methods;

Measurement of pH, mixed liquor suspended solids (MLSS), mixed liquor volatile

suspended solids (MLVSS), chemical oxygen demand (COD) was conducted in

accordance with the standard methods (APHA, 1998). The PNP concentrations were

determined spectrophotometrically using the absorbance values at 400 nm with a

UV/VIS spectrophotometer (GENESYS 20, Thermospectronic). Briefly, sample was

filtered and 1.25 ml 0.1 NaOH was added to each 0.25 ml filtered sample as described

by Chandekar and Ingle, (1990). Growth of the cultures was monitored by measuring

optical density at 600 nm, with a GENESYS, Thermospctronic UV-Vis

spectrophotometer as specified by Ziagova et al., (2007). Specific growth rate was

estimated from the slope of exponential rise phase of growth curve (O.D.goo curve of

SBR) as given by Ziagova et al., (2007).

Percent COD removal and PNP removal was determined using the following

relations:

Influent COD-Effluent COD ^ , ^^ % COD removal = *100

Influent COD

Influent PNP-Effluent PNP ^-.^.^ % PNP removal - • * 100

Influent PNP

For calculating specific degradation rate (SDR) and degradation rate (DR) from the

concentration following formula was used:

Influent PNP-Effluent PNP i'mg) SDR (mg PNP gm VSS"' hr"') = :— 7—-^—-

^ ^ ^ MLVSS tgmjTime(hr)

Influent PNP Cone,-Effluent PNP Cone, fmg/l") DR (mg r' hr ' ) = — • '

Time (hr)

Morphology and surface structure of granules were observed qualitatively with a

scanning electron microscope (STEREO SCAN 360) in accordance with Ivanov et al.,

(2006). Granules were prepared for SEM image by washing with a phosphate buffer

111

• and fixing with 2% glutaraldehyde overnight at 4° C. Fixed granules were washed

with 0.10 M sodium cacodylate buffer, dehydrated by successive passages through

25, 50, 75, 80, 90, 95 and 100% ethanol and dried with a CO2 Critical Point Dryer.

3.2.2 Results and Discussion:

3.2.2.1 Biomass (MLSS and MLVSS) Concentration:

The process of acclimatization involves activation of sludge over a period of 30 days.

This allows the biomass to adapt specifically to PNP. After the acclimatization period

the initial sludge has a MLSS value of 3.1 gm f' and MLVSS value of 1.53 gm f'

while the MLSS and MLVSS concentration was increased to 5.9 gm f' and 3.5 gm f'

at the end of the operation (Fig 3.7) Acclimatized sludge was used as seed sludge for

the cultivation of aerobic granules. It was observed that aerobic granules were small

particles dispersed with the amorphous sludge floes after 18" days of inoculation.

However, after 20" days of inoculation the biomass concentration decreased (MLSS

5.53 to 3.9 gm 1"' and MLVSS from 2.76 to 1.78 gm 1"') due to the reduction in

settling time from 30 to 10 min. The biomass with the smaller settling velocities was

washed out of the reactor gradually. The smaller granules grew rapidly in the

subsequent weeks, while more floc-like sludge were washed out, resulting in the

accumulation of biomass. Similarly on day 40"\ decrease in biomass concentration

(MLSS 5.3 to 3.29 gm f' and MLVSS 2.98 to 1.64 gm r')was observed when settling

time was reduced from 10 to 2 min. It indicates that the selective pressure leads to the

wash out of biomass and also enhanced the growth of granules to form bulk of

biomass in the reactor, as shown by the gradual increase in biomass concentration

beyond 40'' day. The rapid transition of acclimatized activated sludge to aerobic

granules reflected the effectiveness of the operating strategy. The biomass

concentration in the reactor finally showed an upward trend which got stabilized on

58"' day (MLSS value of 5.9 gm 1"' and MLVSS value of 3.5 gm 1"') and remain

constant thereafter.

112

3.2.2.2 Shape of Aerobic Microbial Granules:

Shown in the figure 3.8 a are the microscopic image of the sludge after one week of

inoculation, while Fig 3.8 b & c shows SEM image of mature granules at 4,000 and

15,000 magnifications. Spherical, compact and strong surfaces of stabilized granules

were observed after 60 days. The degradation capability of aerobic granules depends

mainly on their compactness (Khan et al., 2009). Granules with compact structure

have higher resistance to toxic compounds (Glancer et al., 1994; .liang et al., 2002;

Vlami et al., 2005 and Tay et al., 2005). The diameter of the aerobic granules was in

the range of 0.5-1.0 mm and cross-section of the granules shows close packing with

evenly distributed channels which facilitates the movement of the wastewater within

the granules. A close observation of granular surface shows a large diversity of

microbial morphotypes, including bacterial rods and cocci, fungi and diatoms

dispersed in extracellular polymeric substance (EPS). As the microbes are present in

the inner portion of compact granules, so they remain protected from substrates

inhibition affects.

3.2.2.3 Optical Density (O.D.too) Of SBR System:

Cell growth is mentioned in terms of optical density. The O.D.gooof the acclimatized

sludge is around 1.42. Fig 3.9 shows that the O.D.soo of the effluent discharged

decreases from 1.42 to 0.35 and the O.D. oo of SBR increases from 1.54 to 2.53.

Sharp increase in O.D.6ooof the effluent discharge and a sharp decrease in O.D.600 of

SBR at two points were observed due to reduced settling time liom 30 to 10 min (on

day 20" ) and 10 to 2 min (on day 40"'). The trend shown by efiluent O.D.600 was

opposite to the trend observed in case of MLSS. This indicates that effluent O.D.600

was inversely proportional to MLSS concentration and SBR O.D. oo is directly

proportional to MLSS. It is due to the fact that when settling time was reduce

microorganisms with low settling velocity were ultimately washed out of the reactor,

leading to an increase in the O.D.600 of the effluent discharge. Initially the biomass

loss in the effluent discharge was high due to abundance of the microbial floes.

Flowe\'er. decrease in settling time causes the sludge with low settling velocity (loose

^ 113

6 -

5 -

3 -

i 2 n

1 -

MLSS MLVSS

/

/ /

\ / .,.>•>>

!i> : > > >

>. *>

10 —T" 20 30

Days

— I — 40 50 60

Fig 3.7: Variation of MLSS and MLVSS concentration during PNP degradation.

(a)

Fig 3.8 a: Microscopic image of sludge (after one week of inoculation).

114

(b)

(c)

Fig 3.8 b «& c: SEM image of mature granules at 4,000 and 15,000 masnifications.

115

microbial floes) to be washed out and only granules with high settling velocity

(greater than 10.0 m hr'') remained in the reactor. When mature granules were

formed, the O.D.goo of effluent gets stabilized at around 0.35 and SBR stabilized at

2.53 which shows maximum retention of biomass in the SBR and the same can be

used for long term wastewater treatment. Specific growth rate was found to be 0.18

d-'.

3.2.2.4 PNP Removal Profile:

The degradation profile shows that initially the degradation of PNP was high and

afterwards the degradation rate decreases till the end of cycle (Fig 3.10). This is

because in the beginning large amount of PNP was available as a carbon source

(food). Hence, microorganisms react effectively during the first 60 minutes of 240

minutes cycle (with 50% volumetric exchange giving 8 hours HRT) sufficient

amount of PNP was being removed but in next 180 minutes its concentration reduces

as low as 32 mg 1"'. Fig 3.10 also shows that concentration of 40, 80 mg 1"' of PNP

decreases to below detection limit in just 180 minutes but high concentration of PNP

(150. 250, 350 mg 1"') took around 220-230 minutes for removal (greater than 90%).

During the single cycle experiment, PNP removal rate at 40 mg l ' (minimum

concentration) and 350 mg 1"' (maximum concentration treated successfully) was 9.45

mg r' hf' and 83.97 mg l ' hr'' with corresponding removal efficiencies of 94.58%)

and 96%) respectively. It can be concluded that removal rate is directly proportional to

substrate concentration. However, at very high concentration (>350 mg 1"') substrate

inhibition occurs (Kargi and Eker 2005) and removal rate decreases, .lust after

addition of influent, a sharp decrease in PNP concentration was observed (as shown

in Fig 3.10 first 30 minutes), this is due to dilution i.e. when influent containing PNP

added in SBR. then due to presence of wastewater in SBR, PNP concentration

decreases due to dilution.

Degradation profile also gives an idea about the specific degradation rate. Specific

degradation rate increases with PNP concentration and the highest specific

degradation rate was found to be 24 mg PNP gni VSS"' hr''at 350 mg 1"'. However.

116

2.8

2.4

2 . 0 -

g 1-6 Q

•S 1.2 S. O

0.8

0.4

r Effluent I SBR

, / \

• \

1

0 10 20 ' 1 '

30

Davj.

1

40

":- - -

' 1

5 0

- -( -<

1 •)

60

Fig 3.9: Optical Density (O.D.soo) of SBR and effluent during the operation.

Time (min)

Fig 3.10: Concentration profile of PNP during single cycle operation.

117

with further increase in PNP concentration degradation rate decreases due to substrate

inhibition as mentioned above. Yi et al. (2006) reported a maximum specific

degradation rate of 19.3 mg PNP gm VSS"' hr"' at 40 mg of PNP 1"'.

3.2.2.5 COD removal profile:

Similar trends of removal have been followed in this case also. COD removal is a

measure of organic carbon removed from the system. The removal rate of COD was

initially high then shows gradual decrease, with addition of influent in the SBR,

gradual decrease in COD was observed which may be attributed to dilution but due to

microbes action this value of COD further decreases to give COD concentration

below 150 mg f'. Fig 3.11 shows removal efficiency of COD was 92% and 95.32%

at 40 mg r' and 350 mg f' respectively. Fig 3.10 and 3.11 are showing similar trend.

Hence, COD in the system is attributed to the presence of PNP only.

3.2.2.6 Percent Removal Efficiency along with Effluent and Influent COD (mg

Fig 3.12 shows variation of effluent and influent COD along with percent removal

efficiency. The plot shows that initially the COD of effluent was high 254 mg 1"', with

low removal efficiency 51% inspite of low influent COD values because the lack of

compact granules. After few days of cyclic operation compact granules were formed

and system got stable then the COD of influent gradually increased by increasing

PNP concentration but the effluent COD comes out to be quite low.

At the end of cycle intluent. effluent COD and percent removal efficiency were 3168

mg r', 148.15 mg f' and 95.32% respectively. PNP and COD removal efficiencies

96% and 97% at loading rate of 3.85 gm PNP nf" d"' were reported by Sponza et al.,

2005.

118

3500

3000

2500

^2000

g 1500

o o u

1000-

500-

0 -"•>-- - ~(^ -

40mgl

SOmgl

1

-1

150 mg I

250 mg I

350 mg I

50 100 150

Time (min)

I 200 250

Fig 3.11: COD removal profile at different PNP concentration during single cycle

operation.

4000

3500

3000-

2500

2J) 2000-S

Q 1500-1

1000

500

"T < 1 •-

/

I I

/

/

— Influent '- — Effluent

% Removal

/ ' - - , / '•-.A/^'VV\A^ 10

—r-20

— T r -

30

Davs

I 40

—r-50 60

Fig 3.12: COD variation along with % removal efficiency during the study.

119

3.2.2.7 Percent PNP Removal Efficiency along with Effluent and Influent

Concentration:

Fig 3.13 shows the variation of influent and effluent PNP concentrations along with

percent removal efficiency. Fig 3.13 illustrates that initially the concentration of effluent

is high 22.55 mg i"' with low removal efficiency (43.62%) due to the absence of

compact granules although the concentration of influent added was also quite low (40

mg r ). The concentration of effluent did not decreases remarkably in the first few

days of the SBR cycle but after granulation effluent PNP decreases gradually with

corresponding increase in removal efficiency for the rest of the cycle inspite of being

increase in the influent concentration from 40 to 350 mg 1"'. At the end of the process

(350 mg r' PNP influent), final effluent PNP and percent removal efficiencies of 14

mg r ' and 96% were achieved.

3.3.3 Conclusion:

PNP degradation was achieved by aerobic granulation technique in SBR at an organic

load of 0.24 kg PNP m" d"' to 2.1 kg m' d"'. MLSS and MLVSS (biomass) stabilized

at 5.9 gm f' and 3.5 gm r'respectively. Optical density (O.D. oo) of effluent and SBR

got constant 0.35 and 2.53 respectively, which minimize the biomass loss during the

cycle. The graph of influent PNP concentration and COD increases while effluent

remains nearly constant because compact aerobic granules degrade PNP effectively.

PNP removal and COD removal efficiencies observed at the end of operation are 96%)

and 95.32%.

Degradation during single cycle reveals the decreased degradation rate with time. The

perusal of degradation profile illustrates the high removal rate of PNP and COD

during first hour of cycle but later on becomes slow. Removal rate of PNP at 40 mg

r' and 350 mg f' were 9.45 mg f' hf' and 83.97 mg f' hf' along with removal

efficiency of 94.58% and 96% respectively. Removal efficiency of COD at 40 mg

r' (minimum) and 350 mg f' (maximum) were 92% and 95.32% respectively. •

Specific degradation rate was found to be 24 mg PNP gm VSS'' hr"' at 350 mg 1"' and

growth rate was 0.18 d"' observed.

120

350-

„-^ 300 H

^ 250-

1 200 -

C

li 150-

c

u CM 100 -Z 0.

5 0 -

' 1 ' 1

?-:-r-;-3

/

J

v \

1

f

/^

• 1 1 1 1 1

/ •

J

•J

— s — Influent

— —Effluent

% Removal

^^7^^V\ 10 20

—1 r-30

Davs

I 40 50 60

Fig 3,13: Variation of PNP along with % removal efficiency during SBR operation.

121

Aerobic granulation technology is a novel and pronounced biotechnology for

wastewater. This process can effectively be used for treating wastewater, containing

PNP. In this technology the same biomass can be used for a long period of time. High

organic load of PNP as well as the fluctuations in organic load of PNP can be treated

by this process effectively.

122

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133

CHAPTER4

BIODESRADATION OF 2,4-DINITROPHENOL BV AEROBIC

AGGREGATES IN SEQUENCING BATCH REACTOR

Biodegradation of 2,4-Dinitrophenol by Aerobic Aggregates in

Sequencing Batch Reactor

2,4-clinitrophenoI is found in many industries effluent and this effluent wastewater

widely treated by activated sludge process, with higher load of DNP. This process has

negative influence on the biochemical reactions and this decrease the system

treatment efficiency and even system failure.

SBR is proven to degrade the toxic or hard to degrade waste (Herzbrun et al., 1985)

such as DNP. In SBR a higher organic load of DNP can be degraded. In the

degradation of DNP catabolism and anabolism have performed by microbes. Under

favorable conditions the catabolism of microorganisms coupling with anabolism by

energy transfer. This energy used for cell growth and maintenance (Strand et al.,

1999). In other case microbes cannot adjust the all energy produced from catabolism

for cell growth. DNP could dissipate the great mass of energy generated from

catabolism (Strand et al., 1999 and Lin et al.. 2003)

A brief introduction of DNP includes properties, uses and health hazard information

as follows.

4.1 2,4-DINITROPHENOL:

2,4-Dinitrophenol (DNP) is phenolic compound that contains two nitro groups. It is a

cellular metabolic poison.

DNP is a federal hazardous air pollutant and was identified as a toxic air contaminant

in April 1993 under AB 2728. It was registered as a pesticide.

No information about the natural occurrence of DNP was found in the readily-

available literature. Sources of DNP include manufacturing plants, mines, foundries,

metals, petroleum and dye manufacturing plants which use DNP. This is also found in

motor vehicle exhaust.

134

4.1.1 Structure:

In DNP (C6H4N2O5), two ring hydrogen atoms replaced by two nitro goups at ortho

and para position. Molecular structure and resonance structure of DNP are shown in

Fig 4.1 and Fig 4.2.

4.1.2 Appearance:

DNP is a yellow, crystalline solid often found in solution.

4.1.3 Odour: DNP has sweet musty odour.

4.1.4 Physical Properties:

• Physical State at Room Temperature and 1 atm: Solid.

• Molecular Weight: 184.106.

• Boiling Point: 113° C.

• Melting Point: 108° C.

• Relative Density (water = 1): 1.683 g/cm^

• Relative Vapour Density (air = 1): 6.36.

• Vapour Pressure: 2.0* 10"' mm Hg at 25° C.

• Solubility: Water (sparingly), ethyl acetate, acetone, chloroform, pyridine,

carbon tetrachloride, toluene, alcohol, benzene, and aqueous alkaline solutions

(Merck, 1989).

• Solubility in Water: < 1 mg/ ml at 67.1° F (NTP, 1992). DNP and its crystalline

sodium salts are soluble in water and cold water.

• Freezing Point: 235° F = 113° C = 386° K.

• Acidity (pKa): 4.114.

4.1.5 Chemical properties:

4.1.5.1 Reactivity Alert: Special alerts if the chemical is especially reactive.

135

NO:

NO2

Fig4.1: Structure of DNP

Fig 4,2: Resonace structure of DNP

136

• Air and Water Reaction:

Highly flammable and slightly soluble in water.

• Incompatibility:

DNP is incompatible with heavy metals and their compounds. It is also

Incompatible with strong oxidizing agents, strong bases and reducing agents.

DNP reacts with combustibles (NTP, 1992).

• Fire Hazard:

Combustible. It may explode if subjected to heat or flame. Poisonous gas is

produced when heated. Vapours are toxic. Can detonate or explode when

heated under confinement. Forms explosive salts with alkalis and ammonia.

4.1.5.2 Flammability:

DNP is highly flammable.

4.1.5.3 Decomposition:

When heated to decomposition it forms explosive salts with alkalis and ammonia and

emits toxic fumes of nitrogen oxides (Sax, 1989).

4.1.5.4 Hazards:

The primary hazard is Irom blast of an instantaneous explosion and non flying

projectiles and fragments. It is explosive when dry or with less than 15% of water.

4.1.6 Uses:

• It is used in making dyes, wood preservatives, explosives, insecticides,

fungicides and other chemicals, also used as a photographic developer.

• It is used by some bodybuilders and athletes to rapidly lose their body fat.

Overdoses are rarely fatal, but are still reported occasionally. These include

cases of accidental exposure (Leftwich et al., 1982), sujcide (Hsiao et al., 2005

and Hahn et al., 2006) and excessive intentional exposure (Mcfee et al., 2004;

Hahn et al.. 2006 and Miranda et al., 2006).

137

•• In living cells, DNP acts as a proton ionophore, an agent that can shuttle

protons (hydrogen ions) across biological membranes. It defeats the proton

gradient across mitochondria and chloroplast membranes, collapsing the

proton motive force, cell uses to produce most of its ATP chemical energy.

Instead of producing ATP, the energy of the proton gradient is lost as heat.

• DNP is often used in biochemistry research to help explore the bioenergetics

of chemiosmotic and other membrane transport processes.

4.1.7 Health Hazards Information:

The U.S. EPA has established an oral Reference Dose (RfD) of 0.002 mg per kg per

day. The consumption of 0.002 mg per kg or less than this amount over a lifetime

would not result in the chronic and non-cancer effect (U.S. EPA, 1994a). EPA has not

established Reference Concentration (RfC) for DNP (U.S. EPA, 1999). It has been

found in National Priorities List sites identified by the Environmental Protection

Agency (EPA).

4.1.8 Route of Exposure:

Exposure to DNP occurs mainly from breathing, drinking water and eating food that

contains the chemicals.

4.1.9 Health Effects of DNP:

Howard, (1999) had studied the health effects of DNP and reported DNP may cause

methemoglobinemia. At low levels, these chemicals may cause cataracts, serious skin

rashes and decreases in white blood cells. At high levels, this chemical may increase

heart beat and breathing rates and even death.

4.1.9.1 On Animals:

• One study reported an increased incidence of stillborn animals and increased

pup mortality in the offspring of animals exposed to DNP by gavages

(ATSDR. 1995).

138

• According to available animal studies, fatal growth inhibition but no birth

defects were reported in the offspring of animals fed DNP (U.S. EPA, 1994a).

• DNP is considered to have high acute toxicity based on short-term animal

tests in rats and mice (RTECS, 1993).

• One study reported that DNP did not promote tumor development in mice.

(ATSDR, 1995 and HSDB, 1993).

4.1.9.2 On Humans:

• The Reference Dose (0.002 mg per kg per day) is based on cataract formation

in humans. The RfD is an estimate of a daily oral exposure to the human

population without any risk.

• Case reports of women taking DNP orally for weight loss reveals that it may

affect the female reproductive system, but the limited information is

inconclusive. (ATSDR, 1995).

• No information is available on adverse reproductive or developmental effects

of DNP in humans.

• Chronic oral exposure to DNP in humans has caused effects on the bone

marrow, central nervous system (CNS) and cardiovascular system (ATSDR,

1995 and NRC, 1982).

• Acute oral exposure to high levels of DNP in humans has resulted in increased

basal metabolic rate, nausea, vomiting, sweating, dizziness, headache, loss of

weight and other symptoms (ATSDR, 1995 and NRC, 1982).

• Exposure to DNP causes fatigue, tliirst, sweating, headache and weakness. It

may also cause anxiety and excitement.

• DNP has irritating effects on the skin and eyes.

• The substance may cause effects on metabolism, resulting in very high body

temperature. Exposure may result in death.

139

4.1.9.3 Effects on Environment:

Acute toxic effects may include the death of animals, birds, fish and death or low

growth rate in plants. Acute effects are seen after two to four days on animals or

plants come in contact with DNP. DNP may be removed from the atmosphere by

direct photolysis or wet and dry deposition (Atkinson, 1995).

4.1.10 Cancer Risk:

DNP has inherent carcinogenic potential caused by its status as a phenol, production

of free radicals and the release of various compounds stored in adipose tissue stores

during DNP rapid oxidation of fat may also potentially be harmful. DNP has no

carcinogenic effects on humans and animals (ATSDR, 1995; U.S. Department of

Health and Human Services, 1993 and U.S. EPA, 1999). It is also not listed in the

CAPCOA, (1993) as having health values (cancer or non-cancer) for use in risk

assessments.

140

4.2 BIODEGRADATION OF 2,4-DINITROPHENOL IN SEQUENCING

BATCH REACTOR:

Nitrophenols are found in industrial wastes, natural waters, rivers, groundwater, in

pesticides treated soil, agricultural, urban waste and in atmosphere as noxious waste

(Bode et al., 1971; Hallus et al., 1983; Grosjean et al., 1985; Leuenberger et al., 1988

and Rippen et al., 1987). Many nitrophenols and dinitrophenols are listed as "Priority

Pollutants" by the U.S. Environmental Protection Agency and recommends

restricting their concentration in natural waters to below 10 ppm (U.S. environmental

protection Agency, 1976). It is considered to be highly toxic to humans, with a lethal

oral dose of 14 to 43 mg kg'' (National Research Council, 1982). Electrophilic attack

of microorganism is impeded because the addition of one or more nitro groups to the

phenol molecule reduces the electron density of the aromatic ring (Bruhn et al., 1987

and Thiele et al., 1988) and the compound may gather in the environment.

Various records have shown that several variety of organisms can consume mono-

nitrophenols as sources of carbon and energy (Raymond et al., 1971; Spain et al.,

1979; Zeyer et al., 1984 and Zeyer et al., 1986), few researches have reported on

microorganisms that can mineralize dinitrophenols also (Jensen et al., 1967; Zeyer et

al., 1986; Bruhn et al., 1987; Lenke et al., 1992; Sikkema et al., 1994 and Nagal et al.,

1996). DNP is used as a source of carbon and nitrogen by number of organism

shov\ed by many studies in which metobilic pathvv'ay has been described hardly

(Simpson et al., 1953; Gundersen el al., 1956: Germanier et al., 1963; Tewfik et ak,

1966; .lensen et al., 1967; Schmidt et al. 1989; Hess et al.. 1990 and .lensen et al..

1995).

DNP is the most important of the six possible dinitrophenol forms. DNP is familiar

uncoupler of electron transport phosphorylation (Loomis et al., 1948; Simon et al.,

1953 and Ilvicky et al. 1969). DNP is used in the production of sulfur dyes, picric

acid, antiseptics, some pesticides explosive, as an indicator for the detection of

potassium and ammonium ions, photographic developer and wood preservative

141

(Mel'nikov et al.-. 1974), in the medical treatment of obesity and as a pesticide

(Grushko et al., 1982 and The Merck Index, 1989).

DNP caused 'dinitrophenol poisoning' to man exposed to contaminated environment

(Leftwich et al., 1982). This syndrome includes lassitude, malaise, headache,

increased perspiration, thirst, profound weight loss and respiratory failure (Leftwich

et al., 1982). Although DNP is industrially useful but it has to be remove from the

wastewater because of its highly toxic effects.

The earlier methods of biodegradation of the DNP reported are listed in the Table 4.1.

Activated sludge process often exposed to inhibitory pollutants like heavy metals and

toxic organics, with an increasing amount of industrial wastewater effluent. Inhibitory

pollutants have negative effects on biochemical reactions of activated sludge and

accordingly decrease the systems treatment efficiency and cause system failure.

Nitrophenols have higher adverse impact on the respiratory activity of activated

sludge (Tomei et al., 2003 and Ricco et al., 2004). Activated sludge is exposed to

DNP in various municipal wastewater treatment plants because it is used in

manufacture of dye, wood preservative and pesticide (Karim and Gupta, 2006).

Few anaerobic studies have shown that at a high concentration, nitrophenols are not

easily biodegradable and inhibitory to methanogenic microorganisms (Shelton et al.,

1984; Battersby et al., 1989; Oren et al., 1991 and Uberoi et al., 1997). DNP caused

obvious inhibition to methanogenesis (Donlon et al., 1995 and Podeh et al., 1996).

Anaerobic microorganisms in granules were also inhibited by DNP, this even resulted

as complete failure of anaerobic treatment process (Karim and Gupta, 2006).

SBR technology is used for industrial effluent and for domestic wastewater

containing DNP. Preliminary research on DNP degradation in SBR showed that the

rate of degradation could be increased by the addition of glucose as a supplement of

carbon substrate (Hess et al.. 1990). Because of the highly toxic nature of DNP there

is a need to develop a biodegradation method with high organic load. This study was

undertaken for the biodegradation of DNP by microorganisms through sequencing

batch reactor. The main object of this study was to degrade DNP at a high loading

142

Table 4.1 Organic Loading Rate of DNP in Different Mediums:

MEDIUM OBSERVATION REFERENCE

Rhodococcus

koreensis

Mixed species

Mixed species

Rhodococcus

erylhropolis

Mixed species

Mixed species

Mixed species

DNP degradation by free and

immobilized cells were 10.0

and 5.4 nmol min'' per mg cells

and by only immobilized cells

0.45 nmol min'' per mg cells.

The activated sludge acclimated

to 75 mg r' initial DNP had

over 100 times the DNP-

degrading bacteria than an SBR

acclimated to 10 mg 1"' DNP.

Parameter values estimated for

2,4-DNP and 3-NP, were 65.0

and 426.8 mg/gm VSS d for

maximum degradation rates;

475.9 and 7445.7 mgl"'.

Yoon et al., 2000.

Karim et al., 2002.

Wei et al., 2003

Kitovaetal., 2004.

Jo et al., 2004.

Ningthoujam et al.

2005.

She etal., 2005.

143

Mixed species Reinadoetal.,2005.

Mixed species Chen et al., 2006.

Mixed species

Mixed species

Anabaena variahilisshowed a

high ability to remove 2,4-DNP

in the concentration range of 5-

150 )iM under continuous light.

Hikoora et al.

2006.

Tanita et al., 2006.

Rhodococcits opaciis DNP degraded aerobically 0.27

and 0.54 mM within 22 and 28

hours.

Gemini et al., 2007.

Biirkholderia sp. strain

KU46

Hiroaki et al., 2007.

Mixed species

Mixed species

Mixed species

SBR system simultaneously

removed more than 99% of the

NACs at loading rates of 0.36

kgNBm"d"',0.3kg4-NPm"

d"', 0.25 kg AN m"M"'and 0.1

kg 2,4-DNP m-'d"'.

Liu etal.. 2007.

0.56 gm-MLSS gm"' COD and

0.56 d"' values were observed

Reinado et al.,

2008.

Chen et al., 2008.

144

Mixed species

Mixed species

Rhodococcus Opacus

The degradation value was

83.6% when the input power

was 150 W and 60 s was

selected as the discharge time.

All nitrate and 95.5% 2.4-

dinitrophenol could be

simultaneously removed at a

hydraulic retention time of 12 h

in the anaerobic bioreactor.

Zhang et al., 2008.

Zhu et al., 2009.

Weidhaas et al.,

2009.

Mixed species

Rhodococcus

imtechensis Strain

RKJ300

Aragon et al., 2009.

Ghosh etal.. 2010.

145

rate of 0.48 Kg m" d"' and to study the aerobic degradation profile of DNP. The

approach of this study was to degrade DNP over a broad range of concentrations.

4.2.1 Materials and Methods:

4.2.1.1 Reactor Set up and Operation:

Initially sludge acclimatized in aeration tank for 30 days period (Fig 4.3) and then

inoculated into SBR. A column type sequencing aerobic sludge blanket reactor (150

cm high, 5 cm diameter, giving H/D ratio 30) with a working volume of 1.4 1 of

aerobic sludge (total volume of the reactor was 1.5 1) was used. The temperature of

reactor maintains about 30 ±2° C throughout the study. Fine air bubbles were supplied

through an aerator at the reactor bottom at 2.4 1 min"' or 2.7 cm s"' superficial air

velocity.

The reactor was operated sequentially in 6 hours cycles (each consists of 2-30

minutes settling, 5 minutes effluent withdrawal, 5 minutes influent addition, 320-348

minutes aerafion) i.e. 4 cycles of 6 hours each could be performed daily. Fig 4.4 and

Fig 4.5 shows the SBR during aeration and settling, Fig 4.6 shows settled sludge in

SBR after aeration. SBR has two ports at 30 and 70 cm, 30 cm port is used for

collecfing sample for MLSS/MLVSS and periodical sludge wasting. Effluent was

withdrawn Irom port at 70 cm above the bottom with a volumetric exchange ratio of

50% which gives an HRT (hydraulic retention time) of 12 hours. Aerobic granules

started appearing 15"' day. The operation proceeded with abiotic losses of DNP which

were negligible (<5%) under identical operating condition. For abiotic losses, a

control reactor was set up without aerobic sludge having same dimensions and

condifions as experimental reactor.

4.2.1.2 Seed Sludge and Wastewater:

The sludge taken from Star paper mill Saharanpur India, with biomass concentration

(MLSS of 2.5 gm 1"' and MLVSS of 1.33 gm 1"') was acclimatized. Bacterial sludge

had been taken as a source oi'microorganisms, which shorten the acclimatization

146

Fig 4.3: At the time of acclimatization with

DNP in activated sludge process.

m

m

Fig 4.4: SBR while aeration during a

cycle (with DNP).

147

I

#.<tf

Fig 4.5: SBR at the time of settling (with Fig 4.6: Settled sludge after aeration (with

DNP). DNP).

148

period in our case. However for getting more specific microorganisms for DNP

removal, activated sludge was initially conditioned over a 30 days period to allow the

biomass to adapt to DNP before inoculating into SBR. The process was performed for

90 days (30 days acclimatization and 60 days SBR operation) at 30 ±2° C. DNP was

given as sole source of carbon and energy along with macro and micro nutrients (Yi

et al., 2006 and Moy et al., 2002). During acclimatization period 5 mg f' to 20 mg f'

DNP was added along with nutrient solution. In SBR only 20 mg 1"' of DNP was

added in the beginning and its concentration was gradually increased up to 120 mg 1"

with nutrients.

4.2.1.3 Wastewater Feed:

The reactor was fed with synthetic wastewater containing DNP in mineral salt

medium (nutrients solution) with composition given by Yi et al., 2006 (Table. 4.2). A

stock solution of DNP was prepared having concentration of 1 gm 1"' (1,000 mg 1"') in

0.1 N NaOH. The desired concentrations of DNP for experiments were obtained by

proportionate dilution with double distilled water. Stock solution of micronutrients

(Table. 2 Moy et al, 2002) were prepared at a 1000 times concentration. For optimal

microbial growth 1 ml of each was added to the fed solution.

In efOuent v/astewater pH was adjusted to around 7.5 by adding NaHC03 (Jiang et al.,

2002 and Moy et al., 2002) Yang et al.. (2008) reported that a slight low pH favors

fungi dominating granules where as a slight alkaline pH favors bacterial granules. A

slight alkaline pH (around 7.5) is also necessary for proper aerobic granulation and

granulation cease to occur at pH> 8.5 as reported by (Hailei et al., 2006). Temperature

change also affects the reactor performance up to a certain degree. Song et al., (2009)

reported that 30 C is optimum temperature for matured granule cultivation.

4.2.1.4 Analytical Methods:

COD. pH. Temperature, mixed liquor suspended solids (MLSS) and mixed liquor

volatile suspended solids (MLVSS), were determined as specified in APHA, (1998)

149

Table 4.2: Composition of Macronutrients (DNP)

MACRO NUTRIENTS AMOUNT REQUIRED (mg l')

KH2PO4 200

Na2HP04 650

MgS04.7H20 25

FeSO4.7H20' 20

CaCloH.G 30

Table 4.3: Composition of Micronutrients (DNP)

MICRO NUTRIENTS AMOUNT REQUIRED (gm l')

H3BO3 005

ZnCl2 0.05

CuCU 0.03

MnS04.H20 0.05

M07O24.4H2O 0.05

AICI3 0.05

C0CI2.6H2O 0.05

NiCl 0.05

150

Concentration of DNP was determined by UV-Visible spectrophotometer

(SHIMADZU UV mini 1240) at a wavelength maximum (?iniax) of 358 nm, then

converted into concentration with the help of a calibration curve. Samples for

concentration determination were prepared by collecting the effluent and filtering it

and then diluting it before taking absorbance. Optical density (O.D. oo) is measure of

cell growth determined by noting the absorbance of sample (taken from SBR) in a

UV-Visible spectrophotometer (GENESYS, Thermospectronic) at Xmax of 600 nm

using double distilled water as blank as reported by Ziagova et al., (2007). For optical

density, effluent samples were directly taken without filtration. Specific growth rate

was estimated from the slope of exponential rise phase of growth curve (O.D. oo curve

of SBR) as describes by Ziagova et al., (2007). All the experiments were performed

in duplicate.

For calculating specific degradation rate (SDR) and degradation rate (DR) from the

concentration following formula was used:

, ^ , _ Influent DNP-Effluent DNP (mg) SDR(mg DNP gmVSS-'hr-')= , , , , ; c c r ^T- ru ^

MLVSS (gm)Time (hr)

, , Influent DNP Cone-Effluent DNP Cone, (mg/1) DR (mg r' hf') = : -— ^ ^ i ^

^ "' ' Time(hr)

Shape and structure of granules was studied by SEM (scanning electron microscopy)

using STEREOSCAN 360 MICROSCOPE specified by Ivanov et al., (2006).

Granules were prepared for SEM image by washing with a phosphate buffer and

fixing with 2% glutaraldehyde overnight at 4^ C. Fixed granules were washed with

0.10 M sodium cacodylate buffer, dehydrated by successive passages through 25, 50,

75. 80. 90. 95 and 100% ethanol and dried with a CO. Critical Point Dryer.

Percent COD and DNP removal efficiency was calculated by using following these

relations:

Influent COD-Effluent COD , , ^ ^ ' % COD removal = ^ 100

Influent COD

151

Influent DNP-Effluent DNP ^ , „ % DNP removal = —- — " * 100

Influent DNP

4.2.2 F esults and Discussion:

4.2.2.1 Biomass Concentration (MLSS and MLVSS):

After acclimatization period, MLSS and MLVSS of aerobic sludge reached to 3.66

gm r' and 1,94 gm 1'' respectively. The sludge was then inoculated into SBR. Fig 4.7

illustrates that MLSS and MLVSS (biomass concentration in SBR) shows an upward

trend to get stabilized at 6.1 gm 1"' and at 3.98 gm 1"', meanwhile the granules

appeared as major part of sludge. From Fig 4.7, it is also observed that MLSS and

MLVSS decrease at two points because of reduction in settling time (30 to 10 minutes

and 10 to 2 minutes).

Hence, biomass (MLSS and MLVSS) is directly proportional to O.D.goo of SBR.

Settling time was reduced to discard the loose floes and to select compact granules

which are strong enough to withstand the toxic effects of substrates. During the cycle,

compact and stable granules were formed, which were more resistant to DNP. Hence,

increasing concentration did not affect the reactor efficiency. When stable granules

were formed, experiments were performed to study the degradation profile at stable

biomass concentration of MLSS 6.01 gm 1"' and MLVSS 3.98 gm f' at about 30 ±2^ C

temperature and slightly alkaline pH (around 7.5).

4.2.2.2 Shape and Size of Granules:

Fig 4.8 shows stable granules cultivated into the SBR. Fig 4.8 a shows the

microscopic image of sludge after inoculation into SBR for one week, b and c shows

SEM image of granule at 4,000 and 15,000 magnifications. The aggregation of

microbial cells into compact granules served as an effective strategy for protecfion

against ill effects of high,concentrations of DNP. The formation of aerobic granules

consist of five stages: Microbes multiplication phase, fioc appearance phase, floe

152

cohesion phase, mature floe phase and aerobic granule phase (Hailei et al., 2006).

The resistance to toxicity capability of granules depends mainly on their compact

structure (Khan et al., 2009). Granules have high resistance to toxic compounds due

to their compact nature (.liang et al., 2002; Vlamai et al., 2005; Glancer et al., 1994

and Tay et al., 2005). Hence good degradation results were obtained. The diameter of

the aerobic granules was in the range of 1-2 mm. Cross-section of the granules shows

close packing with evenly distributed channels which facilitates the movement of the

wastewater inside the granules. A close observation of granule surface shows a large

diversity of microbial morphotypes, including bacterial rods, cocci, fungi and diatoms

etc.

4.2.2.3 Microbial Density in SBR System (O.D.eoo):

Fig 4.9 shows the microbial density in SBR system in terms of O.D.goo- During

acclimatization the O-D. oo value of SBR increases and gets stabilized at around 1.66

and the value of effluent O.D.(,oo was stabilized at 1.53. When the sludge was

inoculated into SBR, the O.D. ooof the SBR increases gradually and finally stabilized

at around 2.63 but the O.D. oo of the effluent decreases gradually and got stabilized at

0.29. This increase and decrease in O.D.goo of the SBR and effluent respectively, is

accredited to aggregation of loose microbial floes (under the influence of high

hydrodynamic shear force in SBR) to form compact granules which settle fast under

gravity because of their large size.

Initially the settling time was long (30 minutes) which reduces the chances of biomass

washout and cause O.D.600 to increase. On day 20"\ O.D .(,00 was decreased due to

reduction in settling time from 30 to 10 minutes. Again on day 40"'. O.D.500 showed a

similar decreasing trend. This time also the reason behind such behavior is reduction

in settling time from 10 to 2 minutes. Specific growth rate as estimated from the slope

of exponential rise in SBR O.D.500 curve was fbund to be 0.016 d"'.

Due to decrease in settling time, loose microbial floes had been washed out of the

SBR and only thick floes which were compact enough to settle down fast was retained

in the reactor. Such compact floes then gradually transform into microbial

153

s: 5

.2 • * *

55 I..

1: 4 u s: 9

3 -

E .£ 2

1 -

MLSS • ^ I L V S S

/

/

/

/

, • • • / . • • •

, . • • . "

. • •

—1 ' r 20

-< r-50 10 30

Davs

40 60

Fig 4.7: Variation of MLSS and MLVSS concentration of DNP during its

degradation.

(a)

Fig 4.8 a: Microscopic image of sludge (after one weeic of inoculation).

154

dS^£V tm-»y«y af-inao ^ ^ * ' S t ' aai* t w o w ia

gi'.-ifc • • • > ! i^i^'^yj^ I *

(b)

EH7-2^(^>V ' V V 3 ' H 1 ~ - ^s^'f'-U' 'l^.! - T. "Jt fi» fliuiuK^ - Slbf !)4t(. IB r ^ ^ U l Vimti V

(c)

Fig 4.8 b & c: SEM image of mature granules at 4,000 and 15,000 magnifications

155

granules. Since mature granules are compact they settle down easily leading to

retention of biomass. Hence, they can be utilized for long term wastewater treatment.

4.2.2.4 DNP Removal Profile:

The degradation profile shows that initially the degradation rate of DNP was high.

Later on degradation rate decreases till the end of cycle (Fig 4.10). This was due to

the fact that initially microorganisms have large amount of DNP available as their

food, so they degrade it faster in the beginning. During the first 180 minutes of 360

minutes cycle, sufficient amount of DNP was removed. Then in the next 180 minutes,

though its concentration decreases but not at the same rate as before. Fig. 4.10 also

shows that concentration of 20, 40, 60, 80 mg 1"' of DNP was decreased to below

detection limit in just 240 minutes but high concentrafion of DNP (100 and 120 mg

r') took around 300-330 minutes for removal (above 85%). Removal rate and

removal efficiency of DNP were 2.97 mg 1"' hf'and 89% for 20 mg 1"'. For 120 mg f'

removal rate and efficiency were 17.58 mg f' hr"' and 88% of DNP. It is observed that

removal rate was increased as the granules become stable and compact. Specific

degradation rate of DNP was found to be 4.41 mg gm VSS"' hr''. Further rise in DNP

concentration decreased due to substrate inhibition (Kargi and Eker 2005).

4.2.2.5 COD Removal Profile:

The COD removal shows similar trend as DNP removal. The removal rate of COD

was high in the beginning but then goes on decreasing as illustrated in Fig 4.11. Just

after addition of influent in the SBR, there was a sharp decrease in COD which was

attributed to dilution but this value of COD fiirther decreased to give COD

concentration below 120 mg 1"'. The COD was 684 mg 1"' and 4196 mg 1"' at lower (20

mg r') and higher (120 mg I"') load of DNP. The COD removal efficiency was

96.78% and 97.21% respectively. Moreover, the COD removal gives an idea about

the amount of organic carbon removed from the system. From Fig 4.10 and Fig 4.11,

it is clear that DNP and COD removal were in good agreement and directly related to

the performance of reactor.

156

2.8

2 . 4 -

2.0 -

g 1-6-1 a 15 .a 1.2

o 0.8

0 . 4 -

0.0

Effluent » SBR

, * » • »

» ^ j - *

..«*» ,»>**

a » "

» « •

10

/ \ .

20 1

30

Davs

40 50 60

Fig 4.9: Variation of Optical Density (O.D.goo) of SBR system with time.

120

100

M

E • > - •

S3 > O

E it tA a. z Q

80

60

40

2 0 -

20 rag l ' 40 mg I' 60 mg r' 80 mg !' 100 mg r' 120 mg r'

- • I ' I ' 1 I 1 ' 1 1 1 1 1 1 1 r -

-50 0 50 100 150 200 250 300 350 400

Time (min)

Fig 4.10: Concentration profile of DNP during single cycle operation.

157

4.2.2.6 Variation of Influent and Effluent COD along with Percent (%) COD

Removal Efficiency:

Fig 4.12 illustrates that percent COD removal efficiency increases as the operation

proceeds. In the beginning of SBR cycle (at 20 mg 1'') influent and effluent COD

along with removal efficiency were 684 mg 1"', 429 mg 1"' and 37.28% respectively.

At the end of cycle (at 120 mg 1'') influent, effluent and efficiency were 4196 mg \'\

117 mg r' and 97.21% respectively. Efficiency increases with influent DNP

concentration. This may be due to the fact that at high concentration of DNP,

microorganisms react with more efficiency due to availability of large food stock

(since DNP is a carbon source for microorganisms) hence high COD removal

efficiency was achieved. However, with further increase in the amount of DNP,

microbial inhibition occur leading to fall in removal efficiency. Strong and mature

granules can sustain the ill effects of fluctuations in low organic load (684 mg 1"') as

well as high organic load (4196 mg f') which causes the breakdown of conventional

activated sludge process. Hence, aerobic granules cultivated in SBR have proved to

be better and more resistant to toxic xenobiotics than traditional process.

4,2.2.7 Variation of Influent and Effluent DNP Concentration along with Percent

DNP Removal Efficiency:

Similar is the case with influent and effluent concentration together with phenol

removal efficiency. Initially aerobic granulation was not formed, therefore the

effluent concentration was 15.61 mg f' with a removal efficiency of 22%) even though

the influent concentration was quite low (20 mg 1"') This is because instability of the

system towards DNP. But as soon as the system gets stable effluent concentration and

corresponding DNP removal efficiencies were 14.49 mg f'and 88% at 120 mg f'

DNP influent (Fig 4.13).

158

4500 -

4000 -

3500

"— 3000-

E •^ 2500 H

g 2000

u C 1500 Q

y 100O-

500-

0

-50

20 mg I 40 mg r' 60 mg I' 80 mg I' 100 mgl' 120 mgl'

50 100 — I — 150

"T "T 200 250 300 350

Time (min)

400

Fig 4.11;; COD removal profile DNP during degradation in single SBR cycle.

at

o S

a o u

6000

5500

5000

4500

4000 -

3500

3000

2500

2000

1500-

1000-

500

0

/

/

— — Influent —< —Effluent

' % Removal

10 20 30 40

Davs

T ' r 50 60

Fig 4.12: COD variation along with % removal efficiency during the study.

159

_—. '—

E ^ • ^

c +^ SI La S U

e

U

z

^uu -

1 8 0 -

1 6 0 -

1 4 0 -

1 2 0 -

1 0 0 -

8 0 -

6 0 -

4 0 -

2 0 -

0 -

. ._ , . . ^ . . , | - • 1 > 1 • 1 > 1

Wr ^ m * ** ^ '

'•^ "*

•* w

^ * M ^

(- v-v-v-v-v

•#

w

— » — Influent

— —Effluent

^ % Removal

/

V-f-V-Ti'-V

/ t-v-s<-\-v

/

• 1 1 1 • 1 1 1 r 1 • 1 1

10 20 30 40

Davs 50 60

Fig 4.13: Variation of DNP along with % removal efficiency during SBR operation.

160

4.2.2 Conclusion:

This study explored the cultivation of aerobic granules and their role with DNP (20-

120 mg r') as sole carbon and energy source in SBR system. In this work different

DNP concentrations were given to the SBR system with an organic load of COD 684

mg r' to 4196 mg 1"' (organic load of 0.48 kg m" 1"'). Biomass (MLSS and MLVSS)

shows a rising behaviour and stabilizes at 6.1 and 3.98 gm 1''. O.D.600 of SBR and

effluent get stabilized at 2.63 and 0.29. Specific growth rate was found to be 0.016

d"'. DNP and COD removal efficiencies were 88% and 97.21% respectively. Single

cycle operation of SBR reveals a high degradation rate which decreases later. The

inspection of degradation profile illustrates the fast removal rate of DNP and COD

during first hour of cycle but later on becomes slow.

Degradation rate of DNP at 20 mg 1'' was 2.97 mg 1"' hr"' along with 89% removal

efficiency and at 120 mg 1"' degradation rate of DNP wasl7.58 mg 1"' hr"' along with

88% removal efficiency. Removal efficiency of COD at 20 mg f' 96.78% and at 120

mg r' removal efficiency of COD was 97.21%. Specific degradafion rate was 4.41 mg

gmVSS-'hr'.

This study demonstrates that the microbial community in the aerobic granules was

capable of adaptive changes to tolerate the DNP loadings. Hence, aerobic granules

cultivated in SBR have proved to be better and more resistant to DNP than the

traditional process because higher loadings cause the breakdown of conventional

activated sludge process.

161

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LIST OF PUBLICATIONS

1. "Biodegradation of phenol by aerobic granulation technology" Published,

(2009). Water Sci. Technol. 59 (2): 273-278.

2. "Degradation Profile of Phenol in Sequential Batch Reactor" in Press, (2010).

Environ. We Int. J. Sci. Technol. 5: 123-135.

3. "Aerobic Granular Treatment of 2,4-dichiorophenoi" Paper Accepted, (2010).

IJAES.

4. "Aerobic Granulation for Para nitro phenol Biodegradation in a Sequencing

Batch Reactor" Paper Communicated, (2010). Water Res. Management.

5. "Biodegradation of 2,4-dinitrophenol by aerobic granular sludge process"

Paper Communicated, (2010). Environ. Sci. Pollut. Res.

6. "Biodegradation of Para nitro phenol by Aerobic Granulation" Paper in

National Symposium on RTETTB, AMU Aligarh. (2010). pp 29.

7. Abstract on "Biodegradation of 2-Chlorophenol in a sequencing batch reactor"

accepted in conference on Solid Waste Management, Philadelphia, USA.

(2008).

VII

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s- jWA Publishing 2009 Water Science & Technology—WST | 59.2 | 2009

Biodegradation of phenol by aerobic granulation technology

Farah Khan, Mohammad Zain Khan, Shams Qamar Usmani

and Suhail Sabir

ABSTRACT

High organic load and fluctuation in organic load in terms of total phenols is fed into a SBR of

higher height and diameter ratio (H/D 20) and operated at 4 h cycle basis with a volumetric

exchange ratio of 50% resulting in a constant HRT of 8 h. Successful cultivation of aerobic

granules was achieved. Aerobic granulation technology system withstands the inhibitory effect as

well as fluctuation of organic load and a better efficiency as compared to traditional activated

sludge process. Maximum phenol concentration of 650 mgP^ is treated efficiently in 4h which is

equal to an organic load of 3.9kgm ^d '. After granulation the effluent phenol and COD

concentrations were Smgl" ' and 41 mgl"'' respectively. Phenol removal efficiency and COD

removal efficiency of 94% and 95% were achieved. Effluent O.D. oo stabilizes at 0,15 which

corresponds to minimum loss of biomass, which is the main advantage of SBR,

Key words | acclimatization, aerobic degradation, biodegradation, sequential batch reactor (SBR)

Farah Khan Mohammad zain Khan Shams Qamar usmani Suhail sabir (corresponding author) Environmentai Research Laijoratoiy, Department ot cliemistry, Aligarh Muslim university, Aligarh 20200J, India E-mail: drsuhsHssnir^gmail.com

INTRODUCTION

The contamination of water supplies occurs with hazardous

pollutants, the most dangerous and problematic of pollu­

tants are aromatic organic compounds (Atlas 1981; Leahy &

Colwell 1990). These compounds are resistant to degra­

dation because of the staoility of aromatic rings present in

them (Leahy & Colwell 1990). In addition, many aromatic

compounds are toxic at very low concentrations, and many

are believed to be carcinogenic (Dipple 1976; Sims 1980;

National Academy of Sciences 1983).

Phenols and its derivatives are present in the eflluent of

inany industrial processes, among them oil refineries, petro­

chemical plants, coke conversion, pharmaceuticals and

resin industries. F'henols are major environmental pollutants

and found in many industrial waste water due to their wide

spread use. The concentration of phenols as low as 1 mgl"'

are toxic to some acjuatic species (Brown et at. 1967) and at

far lower concentrations causes taste and odor problem

in drinking water. Hence the removal of phenol from waste

water is of obvious interest.

doi: lU,21t)6,'wst,2009.863

Physical, chemical and biological methods are used for

removal of aromatics from the groundwater (Strier 1980;

Atlas 1981: Kobayashi & Rittman 1982; National Academy of

Sciences 1983; Gils & Pirbazari 1986), Among these methods

biological method is preferred if complete oxidation to

carbon dioxide and water can be achieved. Physical and

chemical methods have drawback because in these methods

intermediates are formed, that are even inore toxic than

the original coiripounds (Strier 1980: Gils & Pirbazari

1986). Like in the case of phenol, bulk removal of phenol

by solvent extraction, absorption, chemical oxidation,

incineration and other non biological treatment inethods

are used and these suffer from serious drawback such as

high cost and formation of hazardous by products (Loh

et at. 2000). Biological degradation is generally preferred

due to the lower cost and possibility of complete mineraliz­

ation. I'or many years phenol removal has been carried by

activated sludge system but high phenol loading rate and

fluctuations in phenol loads have been reported to cause

274 F. Khan ef al. \ Biodegraclation of phenol wafer Science & Technology—WST | 59.21 2009

breakdown. of activated sludge system (Wantanabe et al.

1999; Kebret et al. 2000).

Phenol containing waste water is difficult to treat

because of substrate inliibition, whereby microbial growth

and concomitant biodegradation of phenols are hindered by

the toxicity exerted by the substrate itself.

The substrate inhibition difficulties associated with high

strength phenolic waste water can be overcome by

strategies such as cell immobilization (Kenweloh et al.

1989). For example cells of Pseudomonas immobilized in

polysulfones hollow fiber membrane were successful in

degrading phenol at concentration in excess of 500 mgl"',

albeit at relatively slow rate (Loh et al. 2000). Aerobic

granulation represents a relatively new form of cell

immobilization technology that has attracted recent

research attention (Tay et al. 2001, 2002). Aerobic granules

are microbial self-aggregates that are cultivated in Sequen­

cing Aerobic Sludge Blanket Reactors (SBRs) with no need

for inert support for biofilm attachment (Beun et al. 1999).

The compact microbial structure can confer on aerobic

granules the good settleability and the high biodegradation

capacity for toxic and recalcitrant compounds (Jiang & Tay

2002; Tay et al. loo^a; Yi et al. 2006). Aerobic granules have

been successfully cultivated from flocculated activated

sludge fed with phenol as a sole carbon source (Jiang &

Tay 2002). Phenol degrading aerobic granules possessed

high actively compact structure and good settleability, and

kinetics data indicated that these granules have potential to

treat waste water with high phenol loading.

The main objective of this study was to degrade phenol

at a high loading rate of 3.9 Kgrn'^'d^'.

MATERIALS AND METHODS

Reactor set up and operation

A column type sequential aerobic sludge blanket reactor

(5 cm diameter) with working volume 1.41. was used made

of transparent perfex glass. The reactor used was left open

for the growth of natural mixed population. At the bottom

of column was fitted fine bubble for supplying air at

superficial air velocity of 2,67 cm s"', Reactor was operated

sequentially in 4h cycles. To avoid excess loss of sludge, the

reactor was initially operated at high settling time of 30 min

later the settling time was reduced to 10 min and finally to 2 min. At the initial stage feeding time was kept high which was decreased gradually as the process proceeds. The reactor set-up included two sampling port, arranged along the height of column type reactor, one at a distance of 50 cm and, other at 30 cm from the bottom of reactor. The effluent was collected through 50 cm port at a volumetric exchange ratio of 50% giving a hydraulic retention time (HRT) of 8 h. The HRT of 8h was kept constant during the analysis. The use of 30 cm height port was for the collection of MLSS and periodical sludge wasting. The operation proceeded with abiotic loss of phenol in SBR which was negligible under identical operation condition.

Seed sludge and wastewater

The source of aerobic digested sludge with volatile

suspended solid (VSS) content of 5.5gml"' was taken

from secondary clarifier of Star Paper Mill Saharanpur,

India. For the period of first 15 days, it was conditioned in

an aeration tank the content was feed with phenol as a only

source of carbon along with nutrient (Jiang & Tay 2002) and

micro nutrient (Moy et al. 2002). The acclimatization

towards phenol was done in a high precision water bath

at a temperature of 25''''C, After the period of acclimatization

the aeration tank was fed in sequential batch reactor with

synthetic waste water containing phenol which increased

gradually from 50mgl^' to 650 m g l ' in a stepwise process.

At a later stage the acclimatized sludge was inoculated in

SBR at room temperature of 25°C.

Wastewater feed

A stock solution of phenol was prepared having concen­

tration of ]0gmr'(10,000mgr'). The desired con­

centrations of phenol for experiments were obtained

by proportionate dilution with distilled water and fed

in to SBR along with nutrients (NH4CI 0.20gml ';

MgS04.7H20 0.13gmr'; K2HPO4 l.GSgmP' and KH2

PO4 1.35gmr').

Stock solution of micronutrients (H3BO3, 0.05 gmP';

ZnCb, 0.05gmr'; CuCb, 0.03gmr'; MnS04,H20

(NH4)6, O.OSgmr'; M07O24.4H2O, 0.05gmr'; AICI3,

0.05gml ': C0CI2.6H2O, 0.05gmr'; and NiCU,

275 F. Khan e( al \ Biodegradat'cn of phenol Water science & Tcchnology-WST | 59.212009

0.05gml'') were prepared at a 1,000 times concentration.

For optimal microbial growth 1 ml of each was added to the

feed solution. Influent wastewater pH was adjusted to 7 by

adding NaHCOj (Moy et al. 2002).

ANALYTICAL METHODS

Measurement of pH, Suspended solids, Volatile suspended

solids (VSS), Chemical oxygen demand (COD) was con­

ducted in accordance with the Standard Methods (APHA

1998). The phenol concentrations were determined spectro-

photometrically using the absorbance values at 500 nm with

a UV/VIS spectrophotometer (GENESYS 20, Thermospec-

tronic) according to APHA (1995). Percent COD removal

and phenol removal is determined using the following

relations;

% COD removal = [(Influent COD - Effluent COD)/

Influent COD] X 100

7cPhenolremoval = [(Influent cone. - Effluent cone.)/

Influent cone] X 100

Morphology and surface structure of granules were

observed qualitatively with a scanning electron microscope

(STEREO SCAN 360). Granules were prepared for SEM

image by washing with a phosphate buffer and fixing with

2'Vo glutaraidehyde overnight at 4°C. Fixed granules were

washed with 0.10 M sodium cacodylate buffer, dehydrated

by successive passages through 25, 50, 75, 80, 90, 95 and

100% ethanol and dried with a CO2 Critical Point Dryer.

RESULT AND DISCUSSION

Phenols are widely distributed in waste discharge of pulp

and paper industries. So the micro organisms present in

such waste are already acclimatized for phenol and its

derivatives through natural selection. That is why we

choose the aerobic sludge from Star Paper Mill Saharanpur,

INDIA. This already acclimatized sludge reduces condition­

ing period in our case. However for getting more specific

microbes for phenol, sludge was initially acclimatized for

about 15 days to allow biomass to adapt phenol.

The initial sludge has a biomass of 3.5gml"' which

increases 5.2gmP' after conditioning (acclimatization)

period. Then the sludge is inoculated in to SBR for the

cultivation of aerobic granules. Aerobic granules developed

along with amorphous floes were first observed after 10

days of inoculation. However the biomass concentration

reduces to 3.5gmP' due to reduced settling time from

30min to lOmin. The biomass with small settling velocity is

washed out of the reactor. Small granules grew rapidly in

subsequent week, while more floe like sludge washed out

from reactor gradually. The biomass concentration

increases till S.Ggml"' and again shows a sharp decrease,

due to decrease in settling velocity from 10 to 2 min. on day

23. The granules become dominant form of biomass as

evident from gradual increase in biomass concentration

beyond day 24. VSS concentration finally shows an upward

trend and stabilizes at 7.3gml"' (Figure 1).

Stable granules were obtained in the SBR. Stable

granules were compact and strong but not much spherical

this is due to uneven distribution of air as it is supplied

without diffuser. But this distortion in shape of granules

has nothing to do with their degradation efficiency

because high degradation efficiency of granules is only

due to their compact structure. Granules have a high

resistance to toxic xenobiotics due to their compact

structure (Glancer et al. 1994; Jiang & Tay 2002; Tay et al.

2005&; Bergsma-vlami et al. 2005). The diameter of the

u, g

0 5 10 1.1 20 25 . 0 . 5 40 45

Day.s

Figure 1 I Variation of VSS concniitration of phenol (gml ') during its degradation.

276 F. Khan e( al. | Biodegradation of phenol Water Science a Technology—WST | SW12009

1.8

i.6

1.4 •

t '-

O.fi-

0.4-

0.2-

\ \

\ n • v v^

^ ^

0 5 10 15 20 25 30 35 40 45

Days

Figure 3 I Optical density C C ^ o variation with time.

Stabilizes at 0.15. As granules are strong and compact in

structure their settling is faster due to gravity. Tlius, the

effectiveness of the chosen operating strategy was reflected

by decrease in O.D.goo of the effluent (Figure 3).

SBR was initially fed with SOrngT ' of phenol and its

low removal efficiency was observed for the first few days,

but as the granules become stable its removal efficiency

increases and finally 94% removal efficiency is achieved at

the end of the operation. The effluent phenol concentration

was around Trngl"' on day 15 due to wash out of poor

settling floes at a selected pressure, However with increase

in biomass concentration and its adaptation to phenol.

Figure 2 I (3) * (b) SEM image of mature granu)es at 100 and 15,000 magnifications.

granules is about 1 -2 mm (Figure 2a) and (Figure 2b) shows

close pack structure with evenly distributed channels which

facilitates movement of wastewater inside the granules.

Optical density O.D.fioo gives density of the microbes

present in the SBR. The O.D.fioi) of the acclimatized sludge

is around 1.7 which decreases after inoculating in SBR but

shows two sharp increases on day 13 and day 23 due to

reduced settling time from 30 to lOmin and from 10 to

2min. This leads to biomass washout in effluent and hence,

O.D.coo again increases but as granule becomes major part

of reactor, the O.D.ano decreases gradually and finally

8,000

7,0()fl

6,000 -

5,000

a 4,000

g 3.000

2,000 -

1,000

0

• - InnucntCOD r - Efnuont COD • ~ Efllucnl

concentration

V T V ! " "

iv 20 30

Da\'.N

40

Figure 4 I Phenol and COD removal during its degradation.

277 F. Khan e( a/. | Biodegradation of phenol Water Science a Technology—WST | 59.212009

96

94

92

5 9U

88

86

84

- • - COD

* Phenol concentration

10 20 30

Days

40 50

Figure 5 I Percent phenol and COD removal with time.

96

94

-. 92

90

86

84

/

ion 2(K) ."100 400 .'iOO

I'lienol concentration (ni'j 1"')

600 700

Figure 6 I Percent COD removal with concentration of phenol (mgl '),

effluent concentration decreases to 4.9 nig l ' on day 18

resulting in 90.2% removal efficiency, Afterward the

influent concentration was increased stepwise from 50

to 650 from day 18 till end of the operation. Gradual

efficiency for phenol degradation remains unaffected with

the increase in phenol concentration. The average phenol

concentration and removal efficiency was 3 mgl ' (Figure 4)

and 95% (Figure 5) respectively. Stripping of phenol is done

under similar reactor and conditions but without aerobic

sludge. It was found by stripping that abiotic losses for

phenol were \'ery less (<5%).

An organic load of 1.5kgm"^d~' was observed with

the initial influent phenol concentration of about 50mgl"'.

Due to unstability of the system the COD removal efficiency

was observed to be 85% on day 16, which finally becomes

95% (Figure 5). The final COD was 41mgr ' . It wa.s

observed that COD removal efficiency increases with

increase in the phenol concentration reaches a maximum

(95%) at 600mgl'"' of Phenol, further increase in phenol

concentration causes a decrease in COD removal efficiency

(Figure 6) due to microbial inhibition. A slightly alkaline pH

of 7.5-8.5 was maintained throughout the experiment.

CONCLUSION

This study demonstrate that fast settling granules for phenol

degradation can be cultivated in SBR which can tolerate

fluctuation in COD load of 1,000 mgP' and higher COD

load of 7,500 m g r ' . Preconditioned sludge takes less

acclimatization duration because of the presence of already

acclimatized microbes from natural selection. Optical

density O.D.eoo of effluent stabilized at 0.15 which

minimizes the loss of biomass and same microbes can be

used further. Phenol removal and COD removal efficiencies

comes out to be 94% and 95% respectively. Thus the present

method provides technical applicability of SBR for treat­

ment of wastewater containing xenobiotics

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