Application of Plackett- Burman design for the comparative study of the efficiencies of hybrid membrane bioreactors for the treatment of hydrocarbons

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ABSTRACT

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ABSTRACT

In this present work, the effectiveness of pseudomonas and bacillus on degradation of

different oils, especially diesel, petrol, engine oils, etc., were studied. Biological method has

been found to be the harmless method for degrading hydrocarbons with low cost and high

efficiency. Among the biological methods, bioreactor has been the sophisticated method that

can degrade the hydrocarbons to concentrations below the standard limits. Membrane

bioreactors can be broadly defined as systems integrating biological degradation of waste

products with membrane filtration, with good control of biological activity. The effluent from

these bioreactors will be free from chemicals and micro organisms. For the degradation

studies, I developed a media using Plackett-Burman design. It has been found to be the

easiest and time saving procedure for the development of different media with varying

concentrations of nutrients. It offers reduction in BOD within the range of 60-75% and COD

reduction up to 83% within 3 days when introduced into the bioreactor. The temperature was

maintained at 32oC at a pH of 7.

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INTRODUCTION

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INTRODUCTION

The worldwide rise in population and the industrialization during the last few

decades have resulted in ecological unbalance and degradation of the natural resources. One

of the most essential natural resources which have been the worst victim of population

explosion and growing industrialization is water. Today, we withdraw water far faster than it

can be recharged—unsustainably mining what was once a renewable resource (Abramovitz,

1996). Huge quantity of wastewater generated from human settlement and industrial sectors

accompany the disposal system either as municipal wastewater or as industrial wastewater.

This wastewater enriched with varied pollutants and harmful both for human being and the

aquatic flora and fauna, finds its way out into the nearly flowing or stationary water bodies

and thus make the natural sources of water seriously contaminated. The presence of some

harmful pollutants in wastewater deteriorates the water quality considerably and has

damaging effect on both aquatic life and human health (B.C. Meikap, Roy, 1995).

The world’s rapid population growth over the last century has been a major

factor in increasing global water usage. But demand for water is also rising because of

urbanization economic development, and improved living standards. Between 1900 and

1995, for example, global water withdrawals increased by over six times—more than double

the rate of population growth ( Gleick ,1998). In developing countries, water withdrawals are

rising more rapidly—by four percent to eight percent a year for the past decade—also

because of rapid population growth and increasing demand per capita (Marcoux, 1994).

Caught between (a) finite and increasingly polluted water supplies, and (b) rapidly rising

demand from population growth and development, many developing countries face difficult

and uneasy choices. As the World Bank has warned, lack of water is likely to be the major

factor limiting economic development in the decades to come ( Serageldin , 1995).

In recent years, considerable attention has been paid to industrial wastes

discharged to land and surface water. Industrial effluents often contain various toxic metals,

harmful dissolved gases, and several organic and inorganic compounds. Huge quantity of

waste water generated from human settlement and industrial sectors accompany the disposal

system either as municipal wastewater or industrial wastewater (H.M. Jena, et al, 2005). This

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wastewater is enriched with varied pollutants and harmful both to human being and the

aquatic flora and fauna and its successive accumulation in the soil has adverse effect on soil

productivity. All of India’s 14 major rivers are badly polluted. Together they transport 50

million cubic meters of untreated sewage into India’s coastal waters every year. India’s

capital, New Delhi, dumps 200 million liters of raw sewage and 20 million liters of industrial

wastes into the Yamuna River every day as it passes through the city on its way to the Ganges

( Harrison,1992)..

Environmental contamination due to spills and leaks of petroleum

hydrocarbons from storage facilities and distribution systems has resulted in the

contamination of soil and water environments worldwide. Because of the threat they

represent to public health, environmental regulations and the need for the safe use of

renewable and non-renewable resources, multiple clean up strategies for contamination due to

petroleum products have been developed .( Rodríguez-Martínez , 2006).

The exploration, production, refining and distribution of petroleum and

petrochemical products results in the generation of a considerable volume of waste oil

sludges. These sludges come from a variety of sources including storage tank bottoms, oil-

water separators, dissolved air floatation units, cleaning of processing equipment, biological

sludges from waste water treatment units and oil spills in the oil fields, drilling sites and

refineries (Ajay Singh, et al, 2001). The oily sludges are basically composed of oil, water,

solids and their characteristics, such as varied composition, make their reutilization very

difficult, and confer on them high recalcitrance (Ururahy, et al,1999).

A variety of physical, chemical and biological approaches have been

taken to remediating refinery sludges. In many countries these sludges have been

accumulated in large lagoons, facilitating some recycling of oil but requiring later

remediation of residual oily sludges. Attempts to process these sludges using centrifugal

methods to separate oil, water and solids phases is highly capital intensive, is not consistently

effective and still produces residual solids with high petroleum hydrocarbon content.

Another option is to direct oily sludge waste to a delayed coker, however,

this can degrade the sludge quality and reduce its economic value. Foul odors are often

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reported in the coke product after sludge injection and can result in operator complaints.

Sludge injection requires modifications to the coker as well as pretreatment of the sludge.

This pretreatment step requires the use of milling, filtering and centrifugation equipment and

a skilled operator. Thermal desorbers and incinerators have been used for the treatment of

oily sludges. However, most of the above methods are capital intensive and therefore are

associated with overall high sludge treatment costs.(Ajay Singh ,et al,2001).

Microbial degradation of hydrocarbons, through either naturally occurring

processes or engineered systems, has been successfully used to reduce concentrations of these

pollutants to safer environmental levels. Pump and treat systems are one engineering

approach that allows the design of a treatment units for optimum biological operation. When

combined with fixed film microbial growth, such strategies have shown effectiveness in the

processing of sewage and contaminated groundwater. (Enid M. Rodríguez-Martínez, 2006).

Oil effluents from different industries such as refineries, petroleum

treatment plants and different large scale and small scale industries that are dealing with the

petroleum oils contain large amounts of hydrocarbons mainly benzene, toluene, ethyl

benzene, xylene. Most of them are highly water soluble and are toxic. When released into

water sources, they could cause serious consequences in aquatic flora and fauna and can both

directly and indirectly affect humans.

Due to the high energy costs, the potential risk of air pollution and the

persistence of Polycyclic Aromatic Hydrocarbons (PAHs), incineration is not recommended.

Biotreatment can be applied, using the following methods: Composting, Landfarming and

Biopile. All of them exploit the soil biodiversity; however they have the disadvantage of

needing long process times and there is the risk of contaminating air and aquifiers by

leaching. They also demand large areas and are affected by climate. An interesting alternative

to this problem is the use of a bioreactor, since optimum process conditions can be easily

controlled, allowing higher quality final effluent in shorter times (Ururahy,et al,1999).

Several aerobic and anaerobic bioreactors are available for the effective

treatment of hydrocarbons from the petroleum effluents. During the last few years, several

bacterial cultures have been isolated with the ability to degrade the hydrocarbons (Enid M.

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Rodríguez-Martínez, 2006). These bacterial cultures when introduced into suitable bioreactor

with optimum conditions, within a short period degrade the harmful effluents into harmless

products, with concentrations well below the recommended limits (Ururahy,et al,1999). The

treated water from the bioreactor can be either reused or discharged into the water bodies.

MEMBRANE BIOREACTORS

Membrane Bioreactors (MBRs) can be broadly defined as systems integrating

biological degradation of waste products with membrane filtration. They have proven quite

effective in removing organic and inorganic contaminants as well as biological entities from

wastewater. Advantages of the MBR include good control of biological activity, high quality

effluent free of bacteria and pathogens, smaller plant size, and higher organic loading rates

(Cicek,2003). Current applications include water recycling in buildings, municipal

wastewater treatment for small communities, industrial wastewater treatment, and landfill

leachate treatment.

The membrane bioreactor (MBR) process, consisting of an activated sludge

bioreactor and a microfiltration membrane, is an emerging and promising technology

utilizing a biological treatment process. It takes advantage of the rapid development in

membrane manufacturing and has the potential to fundamentally advance the biological

treatment of wastewater. The MBR system has exhibited an excellent effluent quality, a high

biomass concentration without concern for sludge settling problems, a simple flow

configuration, and a small footprint demand. The MBR has been used successfully for

biological treatment of wastewater and for the reclamation of treated effluents (Min Jin, et al,

2005).

ANAEROBIC BIOREACTORS:

As the name indicates, these bioreactors are designed to carry out

biodegradation in the absence of oxygen. The anaerobic process comprises a series of

interdependent phases. Initially complex organic compounds such as lipids, proteins and

carbohydrates, if present, are hydrolyzed to simpler organics. The latter are then fermented to

volatile fatty acids (VFAs) by acidogens .The most common of these fatty acids is ethanoic

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acid. However, propanoic, butanoic and pentanoic acids may also be present in varying

quantities depending on the stability of the process. The acidogens include both facultative

and obligate anaerobic bacteria. Subsequent to the acidogenic phase is the methanogenic

phase. The methanogens are obligate anaerobes and they convert the fatty acids from

acidogenesis to methane and carbon dioxide. This results in substantial decrease in the

organic content of the wastewater. The methane generated offers an avenue for energy

recovery (Buyukgungor, Gurel, 2009).

AEROBIC BIOREACTORS:

The aerobic biodegradation process is represented by the following equation

CxHy + O2 + (microorganisms / nutrient ) ---------- H2O + CO2 + biomass. Aerobic treatment

of waste is the degradation and purification process in which bacteria that thrive in oxygen –

rich environments break down and digest the waste. The mixed aerobic microbial consortium

uses the organic carbon present in the effluent as their carbon and energy source. The

complex organics finally get converted to microbial biomass (sludge) and carbon di oxide

(Behera,2009).

PLACKETT – BURMAN DESIGN:

The efficiency of degradation can be increased several fold by using a media

that supports the growth of the microorganism. The microorganisms belonging to

Pseudomonas species have the ability to use Oil as the sole source of Carbon and energy,

which results in degradation of the oil. When it is supplied with a medium that catalyzes its

growth, the efficiency can be increased by several folds (Allia, et al, 2006).

The cell growth and accumulation of metabolic products in bacteria

are strongly influenced by media composition such as carbon sources, nitrogen sources and

inorganic salts. Thus it is difficult to search for major factors and to optimize them for

biotechnological factors as several factors are involved. The classical method of optimization

involves changing one variable at a time by keeping other factors at fixed levels. Statistical

method for optimization of media effectively tackles the problem, which involves specific

design of experiments which minimizes the error in determining the effect of variables.

Placket-Burman design allows reliable short listing of medium components in fermentation

for further optimization and allows one to obtain unbiased estimates of linear effects of all

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factors with maximum accuracy for given number of observations. (Aravindan Rajendran,et

al,2007).

DEGRADATION OF HYDROCARBONS BY THE MICROORGANISMS

Biodegradation of petroleum hydrocarbons is a complex process that

depends on the nature and on the amount of the hydrocarbons present. Petroleum

hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes

(phenols, fatty acids,ketones, esters, and porphyrins), and the resins (pyridines, quinolines,

carbazoles, sulfoxides and amides).Several factors influence the hydrocarbon degradation.

One of the important factors is their limited availability to the microorganisms.Hydrocarbons

differ in their susceptibility to microbial attack. On the basis of susceptibility to degradation,

the hydrocarbons can be ranked as follows: linear alkanes > branched alkanes > small

aromatics > cyclic alkanes. High molecular weight compounds such as Poly Aromatic

Hydrocarbons (PAHs) are not degraded at all. Bacteria are the microorganisms that are highly

efficient in degrading petroleum hydrocarbons. Bacteria such as Pseudomonas have the

ability to use oil constituents for energy.

FACTORS AFFECTING PETROLEUM HYDROCARBON DEGRADATION

Several factors limit the degradation of hydrocarbons. One among the

important limiting factor is temperature. Decrease in temperature results in lowering the

efficiency of biodegradation. Nutrients are very important ingredients for successful

biodegradation of hydrocarbon pollutants especially nitrogen, phosphorus and in some cases

iron.Another important factor is availability of oxygen. In case of oil spills, the most

important organism capable of degradation is Pseudomonas. Among Pseudomonas sp.,

Pseudomonas putida is the master in degrading oil spills. It has been found that under

aerobic conditions, the efficiency of this organism in degradation is increased several folds.

Hence aerobic treatment is normally preferred for catalyzing the efficiency of this organism.

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REVIEW OF LITERATURE

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BIO-REMEDIATION

Bioremediation is a powerful technical and scientific approach to

alternatively deal with contaminated sites. This process involves the use of microorganisms

to degrade organic pollutants such as hydrocarbons, to concentrations that are undetectable or

below the limits established as safe to all the living organisms and the environment. (Enid M.

Rodríguez-Martínez,2006).

BIO TRANSFORMATION

Biotransformations involve the use of biological agents, in the form of whole

cells or isolated enzymes, to catalyze chemical reactions. Such biotransformation systems

may be used for environmentally benign biocatalysis of synthetic reactions, bioremediation of

pollutants, or waste beneficiation, a combination of these in which the biological agents

convert industrial residues to useful chemical products. In each case, suitable biocatalysts,

and suitable bioreactor systems, each with particular characteristics, are required.(Stephanie

G. Burton,2001)

BIOREACTORS AS USEFUL TOOLS FOR EFFECTIVE ENVIRONMENTAL

REMEDIATION

As waste management practices become more specific, for particular type of

chemical waste, specific treatment system will have to be developed and applied to abate

pollution. In near future, since the regulation concerning discharged wastewater will be

imposed more strictly, an economical, compact and highly efficient wastewater treatment will

be required ( Vijayagopal, Sabarathinam, 2006).

Bioreactors have been commonly developed and implemented for

bioremediation processes. The goal of bioreactor treatment strategies is to optimize

degradation by microbial communities in biofilm or suspended systems in artificially

constructed units that allow tightly controlled growth conditions. In suspended growth

systems, such as activated sludge, or sequencing batch reactors, the contaminated water is

circulated in an aeration basin where microbial populations aerobically degrade the organic

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matter while CO2, H2O and new cells are produced as degradation products. The cells form

sludge, which are settled out in a clarifier unit, and are then either recycled to the aeration

basin or disposed of. (Enid M. Rodríguez-Martínez,2006).

Several aerobic and anaerobic bioreactors are available for the effective

treatment of hydrocarbons from the petroleum effluents. During the last few years, several

bacterial cultures have been isolated with the ability to degrade the hydrocarbons (Enid M.

Rodríguez-Martínez, 2006). These bacterial cultures when introduced into suitable bioreactor

with optimum conditions, within a short period degrade the harmful effluents into harmless

products, with concentrations well below the recommended limits (Ururahy,et al,1999). The

treated water from the bioreactor can be either reused or discharged into the waterbodies

MEMBRANE BIOREACTORS

Membrane Bioreactors (MBRs) can be broadly defined as systems integrating

biological degradation of waste products with membrane filtration. They have proven quite

effective in removing organic and inorganic contaminants as well as biological entities from

wastewater. Advantages of the MBR include good control of biological activity, high quality

effluent free of bacteria and pathogens, smaller plant size, and higher organic loading rates

(Cicek,2003). Current applications include water recycling in buildings, municipal

wastewater treatment for small communities, industrial wastewater treatment, and landfill

leachate treatment.

The membrane bioreactor (MBR) process, consisting of an activated sludge

bioreactor and a microfiltration membrane, is an emerging and promising technology

utilizing a biological treatment process. It takes advantage of the rapid development in

membrane manufacturing and has the potential to fundamentally advance the biological

treatment of wastewater. The MBR system has exhibited an excellent effluent quality, a high

biomass concentration without concern for sludge settling problems, a simple flow

configuration, and a small footprint demand. The MBR has been used successfully for

biological treatment of wastewater and for the reclamation of treated effluents (Min Jin, et al,

2005).

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COMPONENTS OF A MEMBRANE BIOREACTOR:

Membrane bioreactors are composed of two primary parts:

1) The biological unit responsible for the biodegradation of the waste compounds, and

2) The membrane module for the physical separation of the treated water from mixed

liquor.

TYPES OF MEMBRANE BIOREACTORS:

MBR systems can be classified into two major groups according to their

configuration.

1) The first group, which is also commonly known as the integrated MBR, involves outer

skin membranes that are internal to the bioreactor. The driving force across the membrane is

achieved by pressurizing the bioreactor or creating negative pressure on the permeate side of

the membrane. Cleaning of the membrane is achieved through frequent permeate back-

pulsing and occasional chemical backwashing.

2) The second configuration is the recirculated (external) MBR, which involves the

recirculation of the mixed liquor through a membrane module that is outside the bioreactor.

The driving force is the pressure created by high cross-flow velocity along the membrane

surface .(Cicek, 2003).

ANAEROBIC BIOREACTORS:

As the name indicates, these bioreactors are designed to carry out

biodegradation in the absence of oxygen. The anaerobic process comprises a series of

interdependent phases. Initially complex organic compounds such as lipids, proteins and

carbohydrates, if present, are hydrolyzed to simpler organics. The latter are then fermented to

volatile fatty acids (VFAs) by acidogens .The most common of these fatty acids is ethanoic

acid. However, propanoic, butanoic and pentanoic acids may also be present in varying

quantities depending on the stability of the process. The acidogens include both facultative

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and obligate anaerobic bacteria. Subsequent to the acidogenic phase is the methanogenic

phase. The methanogens are obligate anaerobes and they convert the fatty acids from

acidogenesis to methane and carbon dioxide. This results in substantial decrease in the

organic content of the wastewater. The methane generated offers an avenue for energy

recovery (Buyukgungor, Gurel, 2009).

AEROBIC BIOREACTORS:

The aerobic biodegradation process is represented by the following equation

CxHy + O2 + (microorganisms / nutrient ) ---------- H2O + CO2 + biomass. Aerobic treatment

of waste is the degradation and purification process in which bacteria that thrive in oxygen –

rich environments break down and digest the waste. The mixed aerobic microbial consortium

uses the organic carbon present in the effluent as their carbon and energy source. The

complex organics finally get converted to microbial biomass (sludge) and carbon di oxide

(Behera,2009).

DIGESTION PATHWAY

During the oxidation process, continuous contaminants and pollutants are

broken down into end products such as carbon dioxide, water , nitrates, sulphates and

biomass (microorganisms). In the aerobic system, the substrate is used as a source of carbon

and energy.

Synthesis More microorganisms

Waste + Oxygen + Microorganisms

Respiration Energy + End products

It serves as an electron donor, resulting in bacterial growth. The extent of

degradation is correlated with the rate of oxygen consumption in the same substrate. Two

enzymes primarily involved in the process are di and mono oxygenases. The latter enzyme

can act on both aromatic and aliphatic compounds, while the former can act only on aromatic

compounds. Another class of enzymes involved in aerobic condition is peroxidases.

TREATMENT OF OIL SPILLS:

Petroleum based products are the major source of energy for industry and daily

life. Leaks and accidental spills occur regularly during the exploration, production, refining,

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transport and storage of petroleum and petroleum products. The amount of natural crude oil

seepage was estimated to be 600,000 metric tons per year with a range of uncertainty of

200,000 metric tons per year. Release of hydrocarbons into the environment whether

accidental or due to human activities is a main cause of water and soil pollution.

Basically, the oil in the oily wastewater can be classified into three fractions: free oil,

oil/water emulsion and soluble components (Thanh, 2002).

Biodegradation by natural populations of microorganisms represents one of

the primary mechanisms by which petroleum and other hydrocarbon pollutants can be

removed from the environment and is cheaper than other remediation technologies. The

success of oil spill bioremediation depends on one’s ability to establish and maintain

conditions that favor enhanced oil biodegradation rates in the contaminated

environment ( Izanloo, 2007).

One important requirement is the presence of microorganisms with the

appropriate metabolic capabilities. If these microorganisms are present, then optimal rates of

growth and hydrocarbon biodegradation can be sustained by ensuring that adequate

concentrations of nutrients and oxygen are present and that the pH is between 6 and 9. The

physical and chemical characteristics of the oil and oil surface area are also important

determinants of bioremediation success. There are the two main approaches to oil spill

bioremediation : (a) Bioaugmentation, in which known oil-degrading bacteria are added to

supplement the existing microbial population, and (b) Biostimulation, in which the growth of

indigenous oil degraders is stimulated by the addition of nutrients or other growth limiting

co-substrates

PLACKETT-BURMAN DESIGN

The Plackett-Burman Design was developed by R. L.Plackett and

J.P.Burman. Here the design of experiments looks like a matrix, has variables across and runs

down. It is used to study the effects of design parameters on the system states so that

intelligent design decisions can be made. The design was basically meant to improve the

quality control process. It can be used to find out the upper and lower limits of a variable.

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Through this, the quality of a product can be improved in a less expensive way. Overall

quality improvement can save time and money.

The study of factors influencing the production of biomolecules is very

much essential in any bioprocess development. Generally a higher productivity has been

achieved by culture medium optimization. The classical practice of changing one variable at a

time while keeping others at a constant level was found inefficient. This single dimensional

task does not explain interaction effects among the variables and their effect on the

fermentation process. (Aravindan Rajendran,et al,2008).

Moreover it is a time consuming laborious practice because of the large

number of experiments. Conversely, rapid statistical approach enables us to obtain the

physicochemical parameters and factors influencing the fermentation process with limited

number of planned experiments. One such approach is Plackett-Burman design that allows

efficient screening of key variables for further optimization. For the given number of

observation the linear effect of all factors can be screened with maximum accuracy. This

design is practical when investigating large number of factors to produce optimal or near

optimal response.(Aravindan Rajendran,et al,2008).

The cell growth and accumulation of metabolic products in bacteria

are strongly influenced by media composition such as carbon sources, nitrogen sources and

inorganic salts. Thus it is difficult to search for major factors and to optimize them for

biotechnological factors as several factors are involved. The classical method of optimization

involves changing one variable at a time by keeping other factors at fixed levels. Statistical

method for optimization of media effectively tackles the problem, which involves specific

design of experiments which minimizes the the error in determining the effect of variables.

Placket-Burman design allows reliable short listing of medium components in fermentation

for further optimization and allows one to obtain unbiased estimates of linear effects of all

factors with maximum accuracy for given number of observations. (Aravindan Rajendran,et

al,2007).

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AIM:

The present study is focused on the application of Plackett-Burman Design for the

comparative study of the efficiencies of membrane bioreactors for the treatment of

hydrocarbons.

OBJECTIVES OF THE WORK:

Design a Membrane bioreactor incorporating both aerobic and anaerobic phases.

Formulation of a media that enhances the growth and activity of pseudomonas.

Determination of the maximum concentration with which the organism acts.

Comparison of the efficiencies of different types of bioreactors.

Comparison of the efficiencies of different microorganisms.

Measuring the Biochemical Oxygen Demand.

Estimation of the Chemical Oxygen Demand.

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MATERIALS AND METHODOLOGY

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MATERIALS

SAMPLES TAKEN

1) Diesel2) Automobile Engine Oil3) Machine Oil (Heavy Machines)4) Petrol5) Lubrication Oil (Small Machines)

TEST MICROORGANISMS

The bacterial strains used in this study were Pseudomonas Bacillus

CHEMICALS USED

1)FOR PERFORMING PLACKETT- BURMAN DESIGN

a) Magnesium Sulphate

b) Calcium Chloride

c) Di Hydrogen Potassium Phosphate

d) Ammonium Nitrate

e) Ferric Chloride

f) Sodium Chloride

g) Glucose

h) Sodium Carbonate

2)FOR PERFORMING CHEMICAL OXYGEN DEMAND TEST

a) POTASSIUM DICHROMATE SOLUTION (0.1 N)

b) SODIUM THIOSULPHATE(0.1M)

c) SULPHURIC ACID(2M)

d) STARCH SOLUTION

e) POTASSIUM IODIDE (10%)

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3. FOR PERFORMING BIOLOGICAL OXYGEN DEMAND TEST

SODIUMTHIOSULPHATE ( 0.02N)

MANGANESE SULPHATE : 48%

ALKALINE IODINE:

STARCH INDICATOR : 1%

CONCENTRATED SULPHURIC ACID.

GLASSWARES USED

FOR PLACKETT-BURMAN DESIGNBoiling tubes and conical flasks

FOR PERFORMING CODBurette, conical flasks, beakers, pipettes etc.

FOR PERFORMING BODBurette, BOD bottles, conical flasks, beakers, pipettes.

SETTING UP OF BIOREACTORS

Plastic jars Drip bottles Syringes Drip tubes Aerator Air controller Flow regulator Other requirements: glue, packing tape, M-seal.

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METHODOLOGY

1) BIOREACTOR;

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The membrane bioreactor (MBR) was installed in the Biochemistry laboratory of S.B

College, Changanassery. The water sample to be treated during the experiment is prepared by

mixing sterilized water and Oils. .

The MBR consisted of cylindrical bioreactors with a working volume of 5 L and 3 L. The

bioreactors were made of plastic jars. An outlet is fixed 5 cm above the jar’s bottom level.

Similarly, on the top of each bioreactor jar, a hole was made through which the connection

tubes were inserted. The sample water was first stored in the storage tank. From there, the

sample was pumped into the constant water level tank. This tank controlled the influent and

kept the water level in the bioreactor constant as the inflow rate was set by the water level.

The effluent rate of flow was controlled by a flow meter. Aeration pipes were placed in the

jars to provide oxygen for the microorganisms and to generate a shear force which hindered

membrane fouling.

Two membrane bioreactors were used in the experiment, which were operated in a steady

state.

a) Aerobic + Anaerobic MBR

(( ( (Aerobic Tank with

Aerator & Agitator)

Flow Regulator

(Aerobic Tank With

Aerator & Agitator)

Flow Regulator

(Tank for

Back washing) Relesed to Membrane filter

+ Flow regulator Effluent

b) Anaerobic + Aerobic Membrane Bioreactor

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Flow Regulator

Flow Regulator

Flow Regulator

( StorageTank for BackWashing)

Flow Regulator

Effluent

2) PROCEDURE FOR PLACKETT-BURMAN DESIGN

Anaerobic Tank -1

Anaerobic Tank-2

Aerobic Tank with aerator, agitator and membrane

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1) All the chemicals listed in the table were weighed accurately.

2) 12 tubes were arranged serially and labelled as T1-T12 respectively.

3) To each tube, the weighed chemicals were added one by one as shown in the table.

4) The chemicals added were properly mixed by little amount of water and the volume in

all the tubes were made up to 30 ml using distilled water.

5) 30ml of distilled water was poured in another tube labelled as 'c' or control.

6) After mixed properly, the tubes were sterilized in the autoclave.

7) The sterilized tubes were taken out, cooled and to each tube 5% oil was added and

mixed well.

8) All the tubes except the control tube 'C' were inoculated with bacilli.

9) The tubes were then incubated at 37 degrees for 3 days.

10) After 3 days, the absorbance of each sample at 440 nm was estimated

colorimetrically and the percentage of degradation of the oil samples was estimated

by comparing each with a control.

11) Tube labelled T10 showed a higher efficiency.

T10= 83.33%

12) The concentrations of chemicals added in tube T10 were found to be the most efficient.

Trial Level and concentration of variable ( g/L )

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X1

MgSO4

X2

CaCl2

X3

KH2PO4

X4

NH4NO3

X5

FeCl3

X6

NaClX7

GlucoseX8

Na2CO3

T1 0.6 0.2 1 1 0.05 1 3 0.1

T2 0.6 0.6 3 3 0.O5 1 1 0.3

T3 0.2 0.6 1 1 0.15 1 1 0.1

T4 0.6 0.2 3 3 0.05 3 1 0.1

T5 0.6 0.6 3 3 0.15 1 3 0.1

T6 0.6 0.6 1 1 0.15 3 1 0.3

T7 0.2 0.6 1 3 0.05 3 3 0.1

T8 0.2 0.2 1 3 0.15 3 1 0.3

T9 0.2 0.2 3 3 0.15 3 1 0.3

T10 0.6 0.2 3 1 0.15 3 3 0.1

T11 0.2 0.6 3 1 0.05 3 3 0.3

T12 0.2 0.2 3 1 0.05 1 1 0.1

3) THE WINKLER METHOD- Measuring Dissolved Oxygen

PROCEDURE:

a) Carefully fill a 300 ml glass Biological Oxygen Demand stoppered bottle brim- full

with sample water.

b) Immediately add 2ml of manganese sulphate to the collection bottle by inserting the

calibrated pipette just below the surface of the liquid.

c) Add 2ml of alkaline potassium iodide reagent in the same manner.

d) Stopper the bottle with care to be sure no air is introduced. Mix the sample by

inverting several times. Check for air bubbles; discard the sample and start over if any

P a g e | 26

are seen. If oxygen is present, a brownish – orange cloud of precipitate or floc will

appear. When this floc has settle to the bottom, mix the sample by turning it upside

down several times and let it settle again.

e) Add 2ml of concentrated sulphuric acid via a pipette held just above the surface of the

sample. Carefully stopper and invert several times to dissolve the floc. At this point,

the sample is “ fixed” .

f) Add 2ml of starch solution, resulting in blue colour.

g) Continue slowly titrating until the sample turns clear.

h) The concentration of dissolved oxygen in the sample is equivalent to the number of

milliliters of titrant used.

4) PROCEDURE FOR CHEMICAL OXYGEN DEMAND:

a) 50ml of the untreated sample was taken in a dry conical flask.

b) In another conical flask 50ml of distilled water was taken separately.

c) To both of the conical flasks 5ml of potassium dichromate was added. These were then

kept in boiling water bath for 1 hour at 100 degrees.

d) Samples were then taken out and were cooled. To each conical flask 5ml of potassium

iodide and 10ml of sulphuric acid were added. Mixed well.

e) In the mean time a burette was washed, cleaned and was rinsed with sodium

thiosulphate solution. Burette was then filled up to the zero mark with

sodiumthiosulphate solution.

f) The samples in the conical flask were titrated against this sodiumthiosulphate solution

to get a pale yellow colored solution.

g) At this point 2ml of saturated starch solution was added, mixed and was again titrated

until the blue color disappears.

h) This value was note as the COD value of day 1. Similarly the process was repeated after

3 days to get the COD value of day 3. Both the COD values were compared and

substituted in the formula to get the percentage of COD change.

P a g e | 27

RESULT AND DISCUSSION

P a g e | 28

1)PLACKETT- BURMAN DESIGN

The Optical Density measurement was carried out for the media in all the tubes used in

Plackett – Burman design and the values obtained are then compared with that of a Control.

This shows the percentage of degradation of the oil content in each tube.

The percentage of degradation is calculated as follows;

Percentage of degradation = Optical density of untreated sample - Optical density of treated

sample / Optical density of treated sample × 100

The Optical Density readings at 440 nm along with percentage of degradation are represented

as follows;

Test tubes (Serial No)

Control (Untreated sample)

Test (Treated sample)

Percentage of degradation

T1 0.29 0.22 24.14T2 0.04 0.01 75T3 0.06 0.02 75T4 0.04 0.01 75T5 0.05 0.02 60T6 0.23 0.09 39.01T7 0.06 0.02 66.66T8 0.05 0.03 60T9 0.04 0.01 75T10 0.06 0.01 83.33T11 0.05 0.01 80T12 0.05 0.01 80

The media in the tube labelled as T10 shows the highest rate of degradation. It has increased

concentration of Magnesium sulphate, Potassium dihydrogen phosphate, Ferric chloride,

Sodium chloride and Glucose. This clearly indicates that the growth and proliferation of

Pseudomonas is enhanced by these compounds and hence by increasing the concentration of

these compounds, the activity of the organism is enhanced. The Plackett – Burman media,T10,

because of its increased efficiency , was selected for the further degradation studies

P a g e | 29

BOD analysis (Nutrient broth Vs Plackett- Burman Media, T10)Organism: Pseudomonas

BODDay 1 Day 3 D1

( mg /L)D2

( mg /L)Percentage 0f

decrease

D1- D2 /D1 × 100

Nutrient Broth

5.4 2.8 18.09 9.38 48.15

Plackett- Burman

Media, T10

5.4 2.2 18.09 7.37 59.26

D1 - Dissolved Oxygen content of the untreated sample.

D2 – Dissolved Oxygen content of the treated sample.

FORMULA:

Dissolved Oxygen content (D) = K × 200× 0.698 ×Volume of sodium thiosulphate used Volume of sample

Where ‘K’ is a constant with a value of 1.2.

COD analysis (Nutrient broth Vs Plackett- Burman Media, T3 & T10)

CODDay 1 Day 3 Change in

COD(x %)

Percentage 0f decrease in

COD

100 – xNutrient Broth 18.5 14.5 49.23 50.77

Plackett- Burman Media, T10

18.5 13.7 40 60

P a g e | 30

FORMULA FOR CHEMICAL OXYGEN DEMAND:

COD = 8× 0.1(Volume of sample run- Volume of distilled water run) 50

Change in COD = final day COD/ 1st COD ×100

Decrease in COD= 100- Change in COD.

The Biochemical Oxygen Demand (BOD) and the Chemical Oxygen Demand (COD) studies

were carried out using three mediums;

1)Nutrient Broth Media

2) Plackett- Burman Media,T3

The media T10 shows the highest percentage of reduction, both in BOD and COD. Hence it

was selected for the further biodegradation studies .

BOD analysis (5 % Diesel)

Bioreactor Types

Aerobic + Anaerobic membrane Bioreactor &

Anaerobic + Aerobic membrane Bioreactor.

Organism : Pseudomonas

Bioreactor Type

Day 1 Day 3 D1( mg /L)

D2( mg /L)

BOD(mg /L)

Percentage 0f decrease

D1- D2 /D1 × 100

Aerobic + Anaerobic

4.5 1.4 15.08 4.69 10.39 68.89

Anaerobic + Aerobic

4.5 1.6 15.08 5.36 9.72 64.46

P a g e | 31

COD analysis (5 % Diesel)

Bioreactor Types


Anaerobic + Aerobic membrane Bioreactor. Organism : Pseudomonas

PARTICULARSDay

1Day 3 COD

(1st Day)COD

(3rd Day)Change in

COD(x %)


in COD

100 – xAerobic + Anaerobic

18.2 12.2 0.12 0.12 16.67 83.33

Anaerobic + Aerobic

18.2 12.4 0.02 0.03 25 75

The first experiment was carried out in a sample with 5% Diesel. Samples were collected

both before and after treatment. Two types of membrane bioreactors were used;

1) Aerobic + Anaerobic membrane bioreactor.

2) Anaerobic + Aerobic membrane bioreactor.

10 ml of the pseudomonas containing media, T10 was used. The aerobic + anaerobic

membrane bioreactor was found to best with reduction in BOD upto 67.26% and

COD reduction upto 83.33 % within 3 days.

BOD analysis (10 % Diesel)

Bioreactor Types



Bioreactor Type


D2( mg /L)

B0D(mg /L)

Percentage 0f decrease in dissolved oxygen

D1- D2 /D1 × 100

Aerobic + Anaerobic

5.5 1.8 18.42 6.03 12.39 67.26

Anaerobic + Aerobic

5.5 2 18.42 6.70 11.72 63.63

P a g e | 32

COD analysis (10 % Diesel)

Bioreactor Types




CODDay 1 Day 3 COD

( 1st Day)COD

(3rd Day)Change in

COD(x %)


in COD


18.6 12.8 0.13 0.03 23.1 76.9

Anaerobic + Aerobic

18.6 13 0.13 0.04 26.92 73.08

The efficiency of the biodegradation was found to be 76.29% for COD and 67.26% for

BOD within 3 days. Since a fair percent of degradation was obtained, attention was turned

towards the treatment of oil mixture.

BOD analysis (5 % Oil mixture)Bioreactor Types



Bioreactor Type


D2( mg /L)

B0D(mg /L)


D1- D2 /D1 × 100

Aerobic + Anaerobic

5.1 1.7 17.09 5.70 11.39 66.65

Anaerobic + Aerobic

5.1 1.9 17.09 6.37 10.72 62.75

P a g e | 33

COD analysis (5 % Oil mixture )

Bioreactor Types




CODDay 1 Day 3 COD

(1st Day)COD

(3rd Day)Change in

COD(x %)


in COD


18.5 12.6 0.13 0.033 25.85 74.15

Anaerobic + Aerobic

18.5 13 0.13 0.038 29.54 70.46

BOD analysis (5 % Oil mixture)

Bioreactor Types



Organism : Bacillus

Bioreactor Type


D2( mg /L)

B0D(mg /L)


D1- D2 /D1 × 100

Aerobic + Anaerobic

5.1 2 17.09 6.70 10.39 60.08

Anaerobic + Aerobic

5.1 2.3 17.09 7.71 9.38 54.89

P a g e | 34


Bioreactor Types



Organism : Bacillus

CODDay 1 Day 3 COD

(1st Day)COD

(3rd Day)Change in

COD(x %)


in COD


18.5 13.4 0.13 0.045 34.46 65.54

Anaerobic + Aerobic

18.5 13.7 0.13 0.051 39.23 60.77


Bioreactor Types




Bioreactor Type


D2( mg /L)

B0D(mg /L)


D1- D2 /D1 × 100

Aerobic + Anaerobic

6 2.1 20.10 7.04 13.06 64.98

Anaerobic + Aerobic

6 2.3 20.10 7.71 12.39 61.64

P a g e | 35


Bioreactor Types




CODDay 1 Day 3 COD

(1st Day)COD

(3rd Day)Change in

COD(x %)


in COD


19.4 13.2 0.1424 0.043 30.28% 69.72

Anaerobic + Aerobic

19.4 13.5 0.1424 0.048 33.80 66.20


Bioreactor Types



Organism : Bacillus

Bioreactor Type


D2( mg /L)

B0D(mg /L)


D1- D2 /D1 × 100

Aerobic + Anaerobic

6 2.4 20.10 8.05 12.05 59.95

Anaerobic + Aerobic

6 2.8 20.10 9.38 10.72 53.33

P a g e | 36


Bioreactor Types



Organism : Bacillus

CODDay 1 Day 3 COD

(1st Day)COD

(3rd Day)Change in

COD(x %)


in COD


19.4 13.7 0.1424 0.05 35.71 64.29

Anaerobic + Aerobic

19.4 13.5 0.1424 0.056 40 60

The results obtained from the Biochemical Oxygen Demand and the Chemical Oxygen

Demand shows the highest efficiency of degradation of the aerobic + anaerobic membrane

bioreactor, compared with that of anaerobic + aerobic membrane bioreactor. It shows an

efficiency upto 83.33% for COD and BOD reduction upto 68.89%.Pseudomonas was found

to be more effective than Bacillus in degrading hydrocarbons.

P a g e | 37

0

10

20

30

40

50

60

70

Nutrient Broth Plackett Burman Media,T 10

Perc

enta

ge o

f Deg

rada

tion

Plackett - Burman Media Vs Nutrient Broth (Pseudomonas)

BOD

COD

0

10

20

30

40

50

60

70

80

90

Aerobic + Anaerobic Bireactor Anaerobic + Aerobic Bioreactor

Perc

enta

ge o

f D

egra

dati

on

BOD and COD analysis of 5% Diesel (Pseudomonas)

BOD

COD

P a g e | 38

0

10

20

30

40

50

60

70

80

90


Perc

enta

ge o

f Deg

rada

tion

BOD and COD analysis of 10% Diesel (Pseudomonas)

BOD

COD

56

58

60

62

64

66

68

70

72

74

76


Perc

enta

ge o

f Deg

rada

tion

BOD and COD analysis of 5 % Oil mixture (Pseudomonas)

BOD

COD

P a g e | 39

48

50

52

54

56

58

60

62

64

66

68


Perc

enta

ge o

f D

egra

dati

onBOD and COD analysis of 5 % Oil mixture (Bacillus)

BOD

COD

56

58

60

62

64

66

68

70

72


Perc

enta

ge o

f Deg

rada

tion

BOD and COD analysis of 10 % Oil mixture (Pseudomonas)

BOD

COD

P a g e | 40

0

10

20

30

40

50

60

70


Perc

enta

ge o

f Deg

rada

tion

BOD and COD analysis of 10 % Oil mixture (Bacillus)

BOD

COD

Percentage Of Change In BOD & COD

The efficiency of Bioreactors under different conditions such as variations in treatment

phase, concentration of oil content, type of organism used were studied and is tabulated in

percentage as follows

SERIAL NO.

PARTICULARS BOD CODAerobic

+ Anaerobic Bioreactor

Anaerobic + Aerobic

Bioreactor

Aerobic+ Anaerobic Bioreactor

Anaerobic + Aerobic Bioreactor

1 5 % Diesel(Pseudomonas)

68.89 64.46 83.33 75

2 10 % Diesel(Pseudomonas)

67.26 63.63 76.9 73.08

3 5 % oil mixture(Pseudomonas)

66.65 62.75 74.15 70.46

4 5 % oil mixture(Bacillus)

60.08 54.89 65.54 60.77

5 10 % oil mixture(Pseudomonas)

64.98 61.64 69.72 66.20

6 10 % oil mixture(Bacillus)

59.95 53.33 64.29 60

P a g e | 41

The results clearly indicates that the activity and proliferation of the bacteria, Pseudomonas

is greatly increased by the presence of oxygen. After the activation, it can effectively degrade

hydrocarbons and use it for energy and other metabolic activities. This is indicated by the

tests conducted to measure the Biochemical Oxygen Demand and the Chemical Oxygen

Demand. Presence of oxygen is necessary for enhancing the activity of Pseudomonas at the

initaial condition.

P a g e | 42

APPENDIX

P a g e | 43

1 ) TABLE SHOWING THE CONCENTRATIONS OF CHEMICALS USED IN PLACKETT-BURMAN DESIGNTrial Level and concentration of variable ( g/L )

X1

MgSO4

X2

CaCl2

X3

KH2PO4

X4

NH4NO3

X5

FeCl3

X6

NaClX7

GlucoseX8

Na2CO3

T1 0.6 0.2 1 1 0.05 1 3 0.1

T2 0.6 0.6 3 3 0.O5 1 1 0.3

T3 0.2 0.6 1 1 0.15 1 1 0.1

T4 0.6 0.2 3 3 0.05 3 1 0.1

T5 0.6 0.6 3 3 0.15 1 3 0.1

T6 0.6 0.6 1 1 0.15 3 1 0.3

T7 0.2 0.6 1 3 0.05 3 3 0.1

T8 0.2 0.2 1 3 0.15 3 1 0.3

T9 0.2 0.2 3 3 0.15 3 1 0.3

T10 0.6 0.2 3 1 0.15 3 3 0.1

T11 0.2 0.6 3 1 0.05 3 3 0.3

T12 0.2 0.2 3 1 0.05 1 1 0.1

P a g e | 44

2)FOR PERFORMING CHEMICAL OXYGEN DEMAND TEST

POTASSIUM DICHROMATE SOLUTION (0.1 N):

3.676g of potassium dichromate (K2Cr2O7) in 1L distilled water.

SODIUM THIOSULPHATE(0.1M):

5.811g of sodium thiosulphate (Na2S2O3)in 2L of distilled water.

SULPHURIC ACID(2M):

10.8ml of concentrated sulphuric acid (H2SO4) in 100ml of distilled water

STARCH SOLUTION: 0.5g in 50ml distilled water.

POTASSIUM IODIDE (10%): 5 g in 50ml

3. FOR PERFORMING BIOLOGICAL OXYGEN DEMAND TEST

SODIUMTHIOSULPHATE ( 0.02N): 0.49g of sodiumthiosulphate in

100ml distilled water.

MANGANESE SULPHATE : 48%

ALKALINE IODINE:

7.5g of potassium iodide in 70% potassium hydroxide.

STARCH INDICATOR : 1%

CONCENTRATED SULPHURIC ACID.

P a g e | 45

Conclusion

CONCLUSION

P a g e | 46

In this study a media formulated by Plackett- Burman design,T10 was found as a suitable

medium for the growth and activity of pseudomonas. This media was used in the Membrane

Bioreactors for the treatment of water samples by pseudomonas.. T10 media provided the

entire essential nutrients for the organism so that it can grow and degrade oils efficiently.

Under this study a lab scale Membrane bioreactor was designed and was used to compare the

efficiency of various aerobic- anaerobic bioreactors in the treatment of oils. A comparative

study of the efficiencies of the organisms, Pseudomonas and Bacillus, in the degradation of

the hydrocarbon content of the oil samples, under different conditions (Aerobic + Anaerobic

and Anaerobic + Aerobic) were carried out. It was found that the aerobic + anaerobic

bioreactor was the best with 83.33% reduction in COD and more than 60% reduction in

BOD. In both these cases, Pseudomonas, was found to be the best, since it depends the oils

for its energy and growth. Hence it can degrade the oil content far faster and better than

bacilli and it is indicated accurately by the Biochemical Oxygen Demand Test and the

Chemical Oxygen Demand Test.

P a g e | 47

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P a g e | 48

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Documents

Application of Plackett- Burman design for the comparative study of the efficiencies of hybrid membrane bioreactors for the treatment of hydrocarbons